Proteinase k resistant surface protein of neisseria meningitidis

ABSTRACT

The identification of a highly conserved, immunologically accessible antigen at the surface of  Neisseria  facilitates treatment, prophylaxis, and diagnosis of  Neisseria  diseases. This antigen is highly resistant to Proteinase K and has an apparent molecular weight of 22 kDa on SDS-PAGE. Specific polynucleotides encoding proteins of this class have been isolated from three  Neisseria meningitidis  strains and from one  Neisseria gonorrhoeae  strain. These polynucleotides have been sequenced, and the corresponding full-length amino acid sequences of the encoded polypeptides have been deduced. Recombinant DNA methods for the production of the  Neisseria  surface protein, and antibodies that bind to this protein are also disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/582,527 filed Oct. 16, 2006, which is a continuation of U.S. patentapplication Ser. No. 09/684,883, filed Oct. 6, 2000, now issued as U.S.Pat. No. 7,273,611 on Sep. 25, 2007; which is a continuation applicationof U.S. patent application Ser. No. 08/913,362, filed Nov. 13, 1997, nowissued as U.S. Pat. No. 6,287,574; which is the National Stage ofInternational Application No. PCT/CA96/00157, filed Mar. 15, 1996, whichis a continuation of U.S. patent application Ser. No. 08/406,362, filedMar. 17, 1995, now abandoned, and which claims the benefit of U.S.Provisional Patent Application No. 60/001,983, filed Aug. 4, 1995, allof which are incorporated herein by reference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 484112_(—)417C6—SEQUENCE_LISTING.txt. The textfile is 25 KB, was created on Oct. 30, 2007, and is being submittedelectronically via EFS-Web.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a highly conserved, immunologically accessibleantigen at the surface of Neisseria meningitidis organisms. This uniqueantigen provides the basis for new immunotherapeutic, prophylactic anddiagnostic agents useful in the treatment, prevention and diagnosis ofNeisseria meningitidis diseases. More particularly, this inventionrelates to a proteinase K resistant Neisseria meningitidis surfaceprotein having an apparent molecular weight of 22 kDa, the correspondingnucleotide and derived amino acid sequences (SEQ ID NO:1 to SEQ IDNO:26), recombinant DNA methods for the production of the Neisseriameningitidis 22 kDa surface protein, antibodies that bind to theNeisseria meningitidis 22 kDa surface protein and methods andcompositions for the diagnosis, treatment and prevention of Neisseriameningitidis diseases.

2. Description of the Related Art

Neisseria meningitidis is a major cause of death and morbiditythroughout the world. Neisseria meningitidis causes both endemic andepidemic diseases, principally meningitis and meningococcemia [Gold,Evolution of meningococcal disease, p. 69, Vedros N. A., CRC Press(1987); Schwartz et al., Clin. Microbiol. Rev., 2, p. S118 (1989)]. Infact, this organism is one of the most common causes, after Haemophilusinfluenzae type b, of bacterial meningitis in the United States,accounting for approximately 20% of all cases. It has been welldocumented that serum bactericidal activity is the major defensemechanism against Neisseria meningitidis and that protection againstinvasion by the bacteria correlates with the presence in the serum ofanti-meningococcal antibodies [Goldschneider et al., J. Exp. Med. 129,p. 1307 (1969); Goldschneider et al., J. Exp. Med., 129, p. 1327(1969)].

Neisseria meningitidis are subdivided into serological groups accordingto the presence of capsular antigens. Currently, 12 serogroups arerecognized, but serogroups A, B, C, Y, and W-135 are most commonlyfound. Within serogroups, serotypes, subtypes and immunotypes can beidentified on outer membrane proteins and lipopolysaccharide [Frasch etal., Rev. infect. Dis. 7, p. 504 (1985)].

The capsular polysaccharide vaccines presently available are noteffective against all Neisseria meningitidis isolates and do noteffectively induce the production of protective antibodies in younginfants (Frasch, Clin. Microbiol. Rev. 2, p. S134 (1989); Reingold etal., Lancet, p. 114 (1985); Zollinger, in Woodrow and Levine, Newgeneration vaccines, p. 325, Marcel Dekker Inc. N.Y. (1990)]. Thecapsular polysaccharide of serogroups A, C, Y and W-135 are presentlyused in vaccines against this organism. These polysaccharide vaccinesare effective in the short term, however the vaccinated subjects do notdevelop an immunological memory, so they must be revaccinated within athree-year period to maintain their level of resistance.

Furthermore, these polysaccharide vaccines do not induce sufficientlevels of bactericidal antibodies to obtain the desired protection inchildren under two years of age, who are the principal victims of thisdisease. No effective vaccine against serogroup B isolates is presentlyavailable even though these organisms are one of the primary causes ofmeningococcal diseases in developed countries. Indeed, the serogroup Bpolysaccharide is not a good immunogen, inducing only a poor response ofIgM of low specificity which is not protective [Gotschlich et al., J.Exp. Med., p. 129, 1349 (1969); Skevakis et al., J. Infect. Dis., 149,p. 387 (1984); Zollinger et al., J. Clin. Invest., 63, p. 836 (1979)].Furthermore, the presence of closely similar, crossreactive structuresin the glycoproteins of neonatal human brain tissue [Finne et al.,Lancet, p. 355 (1983)] might discourage attempts at improving theimmunogenicity of serogroup B polysaccharide.

To obtain a more effective vaccine, other Neisseria meningitidis surfaceantigens such as lipopolysaccharide, pili proteins and proteins presentin the outer membrane are under investigation. The presence of a humanimmune response and bactericidal antibodies against certain of theseproteinaceous surface antigens in the sera of immunized volunteers andconvalescent patients was demonstrated [Mandrell and Zollinger, Infect.Immun., 57, p. 1590 (1989); Poolman et al., Infect. Immun., 40, p. 398(1983); Rosenquist et al., J. Clin. Microbiol., 26, p. 1543 (1988);Wedege and Froholm, Infect. Immun. 51, p. 571 (1986); Wedege andMichaelsen, J. Clin. Microbiol., 25, p. 1349 (1987)].

Furthermore, monoclonal antibodies directed against outer membraneproteins, such as class 1, ⅔ and 5, were also reported to bebactericidal and to protect against experimental infections in animals[Brodeur et al., Infec. Immun., 50, p. 510 (1985); Frasch et al, Clin.Invest. Med., 9, p. 101 (1986); Saukkonen et al. Microb. Pathogen., 3,p. 261 (1987); Saukkonen et al., Vaccine, 7, p. 325 (1989)].

Antigen preparations based on Neisseria meningitidis outer membraneproteins have demonstrated immunogenic effects in animals and in humansand some of them have been tested in clinical trials [Bjune et al.,Lancet, p. 1093 (1991); Costa et al., NIPH Annals, 14, p. 215 (1991);Frasch et al., Med. Trop., 43, p. 177 (1982); Frasch et al., Eur. J.Clin. Microbiol., 4, p. 533 (1985); Frasch et al. in Robbins, BacterialVaccines, p. 262, Praeger Publications, N.Y. (1987); Prasch et al, J.Infect. Dis., 158, p. 710 (1988); Moreno et al. Infec. Immun., 47, p.527 (1985); Rosenqvist et al., J. Clin. Microbiol., 26, p. 1543 (1988);Sierra et al., NIPH Annals, 14, p. 195 (1991); Wedege and Froholm,Infec. Immun. 51, p. 571 (1986); Wedege and Michaelsen, J. Clin.Microbiol., 25, p. 1349 (1987); Zollinger et al., J. Clin. Invest., 63,p. 836 (1979); Zollinger et al., NIPH Annals, 14, p. 211 (1991)].However, the existence of great interstrain antigenic variability in theouter membrane proteins can limit their use in vaccines [Frasch, Clin.Microb., Rev. 2, p. S134 (1989)]. Indeed, most of these preparationsinduced bactericidal antibodies that were restricted to the same orrelated serotype from which the antigen was extracted [Zollinger inWoodrow and Levine, New Generation Vaccines, p. 325, Marcel Dekker Inc.N.Y. (1990)]. Furthermore, the protection conferred by these vaccines inyoung children has yet to be clearly established. The highly conservedNeisseria meningitidis outer membrane proteins such as the class 4[Munkley et al. Microb. Pathogen., 11, p. 447 (1991)] and the lipprotein (also called H.8) [Woods et al., Infect. Immun., 55, p. 1927(1987)] are not interesting vaccine candidates since they do not elicitthe production of bactericidal antibodies. To improve these vaccinepreparations, there is a need for highly conserved proteins that wouldbe present at the surface of all Neisseria meningitidis strains and thatwould be capable of eliciting bactericidal antibodies in order todevelop a broad spectrum vaccine.

The current laboratory diagnosis of Neisseria meningitidis is usuallydone by techniques such as Gram stain of smear preparations, latexagglutination or coagglutination, and the culture and isolation onenriched and selective media [Morello et al., in Balows, Manual ofClinical Microbiology, p. 258, American Society for Microbiology,Washington (1991)]. Carbohydrate degradation tests are usually performedto confirm the identification of Neisseria meningitidis isolates. Mostof the described procedures are time-consuming processes requiringtrained personnel. Commercial agglutination or coagglutination kitscontaining polyvalent sera directed against the capsular antigensexpressed by the most prevalent serogroups are used for the rapididentification of Neisseria meningitidis. However, these polyvalent seraoften nonspecifically cross-react with other bacterial species and forthat reason should always be used in conjunction with Gram stain andculture. Accordingly, there is a need for efficient alternatives tothese diagnostic assays that will improve the rapidity and reliabilityof the diagnosis.

BRIEF SUMMARY OF THE INVENTION

The present invention solves the problems referred to above by providinga highly conserved, immunologically accessible antigen at the surface ofNeisseria meningitidis organisms. Also provided are recombinant DNAmolecules that code for the foregoing antigen, unicellular hoststransformed with those DNA molecules, and a process for makingsubstantially pure, recombinant antigen. Also provided are antibodiesspecific to the foregoing Neisseria meningitidis antigen. The antigenand antibodies of this invention provide the basis for unique methodsand pharmaceutical compositions for the detection, prevention andtreatment of Neisseria meningitidis diseases.

The preferred antigen is the Neisseria meningitidis 22 kDa surfaceprotein, including fragments, analogues and derivatives thereof. Thepreferred antibodies are the Me-1 and Me-7 monoclonal antibodiesspecific to the Neisseria meningitidis 22 kDa surface protein. Theseantibodies are highly bacteriolytic against Neisseria meningitidis andpassively protect mice against experimental infection.

The present invention further provides methods for isolating novelNeisseria meningitidis surface antigens and antibodies specific thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C depicts the nucleotide and derived amino acid sequences ofthe Neisseria meningitidis strain 608B 22 kDa surface protein (SEQ IDNO:1; SEQ ID NO:2). Conventional three letter symbols are used for theamino acid residues. The open reading frame extends from the start codonat base 143 to the stop codon at base 667. The box indicates theputative ribosome binding site whereas the putative −10 promotersequence is underlined. A 19-amino-acid signal peptide is alsounderlined.

FIG. 2 is a photograph of a Coomassie Blue stained 14% SDS-PAGE geldisplaying a-chymotrypsin and trypsin digests of Neisseria meningitidisstrain 608B (B:2a:P1.2) outer membrane preparations. Lane 1 contains thefollowing molecular weight markers: Phosphorylase b (97,400); bovineserum albumin (66,200); ovalbumin (45,000); carbonic anhydrase (31,000);soybean trypsin inhibitor (21,500); and lysozyme (14,400). Lane 2contains undigested control outer membrane preparation. Lane 3 containsa-chymotrypsin treated preparation (2 mg of enzyme per mg of protein);lane 4 contains trypsin treated preparation.

FIG. 3 a is a photograph of a Coommasie Blue stained 14% SDS-PAGE geldisplaying proteinase K digests of Neisseria meningitidis strain 608B(B:2a:P1.2) outer membrane preparations. Lanes 1, 3, 5, and 7 containundigested control. Lanes 2, 4, 6 and 8 contain outer membranepreparations digested with proteinase K (2 IU per mg of protein). Lanes1 to 4 contain preparations treated at pH 7.2. Lanes 5 to 8 containpreparations treated at pH 9.0. Lanes 1, 2, 5 and 6 contain preparationstreated without SDS. Lanes 3, 4, 7 and 8 contain preparations treated inthe presence of SDS. Molecular weight markers are indicated on the left(in kilodaltons).

FIG. 3 b is a photograph of a Coomassie Blue stained 14% SDS-PAGE geldisplaying the migration profiles of affinity purified recombinant 22kDa protein. Lane 1 contains molecular weight markers: Phosphorylase b(97,400), bovine serum albumin (66,200), ovalbumin (45,000), carbonicanhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme(14,400). Lane 2 contains 5 μg of control affinity purified recombinant22 kDa protein. Lane 3 contains 5 μg of affinity purified recombinant 22kDa protein heated at 100° C. for 5 min. Lane 4 contains 5 μg ofaffinity purified recombinant 22 kDa protein heated at 100° C. for 10min. Lane 5 contains 5 μg of affinity purified recombinant 22 kDaprotein heated at 100° C. for 15 min.

FIGS. 4A & 4B are photographs of Coomassie Blue stained 14% SDS-PAGEgels and their corresponding Western immunoblots showing the reactivityof monoclonal antibodies specific to the Neisseria meningitidis 22 kDasurface protein. Outer membrane preparation from Neisseria meningitidisstrain 608B (B:2a:P1.2) (A) untreated; (B) Proteinase K treated (2 IUper mg of protein). Lane 1 contains molecular weight markers andcharacteristic migration profile on 14% SDS-PAGE gel of outer membranepreparations. Lane 2 contains Me-2; Lane 3 contains Me-3; lane 4contains Me-5; lane 5 contains Me-7; and lane 6 contains an unrelatedcontrol monoclonal antibody. The molecular weight markers arephosphorylase b (97,400), bovine serum albumin (66,200), ovalbumin(45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor(21,500) and lysozyme (14,400). The immunoblot results shown in FIG. 4for Me-2, Me-3, Me-5, Me-6 and Me-7 are consistent with the immunoblotresults obtained for Me-1.

FIG. 5 is a graphic depiction of the binding activity of the monoclonalantibodies to intact bacterial cells. The results for representativemonoclonal antibodies Me-5 and Me-7 are presented in counts per minute(“CPM”) on the vertical axis. The various bacterial strains used in theassay are shown on the horizontal axis. A Haemophilus influenzaeporin-specific monoclonal antibody was used as a negative control.Background counts below 500 CPM were recorded and were subtracted fromthe binding values.

FIGS. 6A-6C are photographs of stained 14% SDS-PAGE gels and theircorresponding Western immunoblot demonstrating the purification of therecombinant 22 kDa Neisseria meningitidis surface protein fromconcentrated culture supernatant of Escherichia coli strain BL21 (DE3).FIG. 6(A) is a photograph of a Coomassie Blue and silver stained 14%SDS-Page gel. Lane 1 contains the following molecular weight markers:phosphorylase b (97,400), bovine serum albumin (66,200), ovalbumin(45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor(21,500) and lysozyme (14,400). Lane 2 contains outer membrane proteinpreparation extracted from Neisseria meningitidis strain 608B (serotypeB:2a:p1.2)(10 mg). Lane 3 contains concentrated culture supernatant ofEscherichia coli BL21 (DE3) (10 mg). Lane 4 contains affinity purifiedrecombinant 22 kDa Neisseria meningitidis surface protein (1 mg). FIG.6(B) is a photograph of a Coomassie Blue stained 14% SDS-PAGE gel ofa-chymotrypsin, trypsin and proteinase K digests of affinity purifiedrecombinant 22 kDa Neisseria meningitidis surface protein. Lane 1contains the following molecular weight markers: phosphorylase b(97,400), bovine serum albumin (66,200), ovalbumin (45,000), carbonicanhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme(14,400). Lanes 2 to 5 contain purified recombinant 22 kDa Neisseriameningitidis surface protein (2 mg). Lanes 7 to 10 contain bovine serumalbumin (2 mg). Lanes 2 and 7 contain undigested protein (“NT”). Lanes 3and 8 contain α-chymotrypsin (“C”) treated protein (2 mg of enzyme permg of protein). Lanes 4 and 9 contain trypsin (“T”) treated protein (2mg of enzyme per mg of protein). Lanes 5 and 10 contain proteinase K(“K”) treated protein (2 IU per mg of protein). FIG. 6(C) is aphotograph of the Western immunoblotting of a duplicate gel usingmonoclonal antibody Me-5.

FIG. 7 is a graphical depiction of the bactericidal activity of proteinA-purified anti-Neisseria meningitidis 22 kDa surface protein monoclonalantibodies against Neisseria meningitidis strain 608B (B:2a:P1.2). Thevertical axis of the graph shows the percentage of survival of theNeisseria meningitidis bacteria after exposure to various concentrationsof monoclonal antibody (shown on the horizontal axis of the graph).

FIG. 8A-8B depicts the nucleotide and derived amino acid sequences ofthe Neisseria meningitidis strain MCH88 22 kDa surface protein (SEQ IDNO:3; SEQ ID NO:4). Conventional three letter symbols are used for theamino acid residues. The open reading frame extends from the start codonat base 116 to the stop codon at base 643.

FIG. 9A-9B depicts the nucleotide and derived amino acid sequences ofthe Neisseria meningitidis strain Z4063 22 kDa surface protein (SEQ IDNO:5; SEQ ID NO:6). Conventional three letter symbols are used for theamino acid residues. The open reading frame extends from the start codonat base 208 to the stop codon at base 732.

FIG. 10A-10B depicts the nucleotide and derived amino acid sequences ofthe Neisseria gonorrhoeae strain b2, 22 kDa surface protein (SEQ IDNO:7; SEQ ID NO:8). Conventional three letter symbols are used for theamino acid residues. The open reading frame extends from the start codonat base 241 to the stop codon at base 765.

FIG. 11A-11C depicts the consensus sequence (SEQ ID NO:29) establishedfrom the DNA sequences of the four strains of Neisseria and indicatesthe substitutions or insertion of nucleotides specific to each strain.

FIG. 12 depicts the consensus sequence (SEQ ID NO:30) established fromthe protein sequences of the four strains of Neisseria and indicates thesubstitutions or insertion of amino acid residues specific to eachstrain.

FIG. 13 represents the time course of the immune response to therecombinant 22 kDa protein in rabbits expressed as the reciprocal ELISAtiter. The rabbits were injected with outer membrane preparations fromE. coli strain JM109 with plasmid pN₂₂O₂ or with control plasmid pWKS30.The development of the specific humoral response was analyzed by ELISAusing outer membrane preparations obtained from Neisseria meningitidisstrain 608B (B:2a:P1.2) as coating antigen.

FIG. 14 represents the time course of the immune response to therecombinant 22 kDa protein in Macaca fascicularis (cynomolgus) monkeysexpressed as the reciprocal ELISA titer. The two monkeys wererespectively immunized with 100 μg (K28) and 200 μg (1276) of affinitypurified 22 kDa protein per injection. The control monkey (K65) wasimmunized with 150 μg of unrelated recombinant protein following thesame procedure. The development of the specific humoral response wasanalyzed by ELISA using outer membrane preparations obtained fromNeisseria meningitidis strain 608B (B:2a:P1.2) as coating antigen.

FIG. 15 is a graphic representation of the synthetic peptides of theinvention (SEQ ID NO:2) as well as their respective position in the full22 kDa protein of Neisseria meningitidis strain 608B (B:2a:P1.2).

FIG. 16 is a map of plasmid pNP2204 containing the gene which encodesthe Neisseria meningitidis 22 kDa surface protein 22 kDa, Neisseriameningitidis 22 kDa surface protein gene; Ampi^(R),ampicillin-resistance coding region; ColE1, origin of replication;cl857, bacteriophage A cl857 temperature-sensitive repressor gene; λPL,bacteriophage λ transcription promoter; T1 transcription terminator. Thedirection of transcription is indicated by the arrows. BglIII and BamH1are the restriction sites used to insert the 22 kDa gene in the p629plasmid.

DETAILED DESCRIPTION OF THE INVENTION

During our study of the ultrastructure of the outer membrane ofNeisseria meningitidis we identified a new low molecular weight proteinof 22 kilodaltons which has very unique properties. This outer membraneprotein is highly resistant to extensive treatments with proteolyticenzymes, such as proteinase K, a serine protease derived from the moldTritirachium album limber. This is very surprising since proteinase Kresistant proteins are very rare in nature because of the potency, widepH optimum, and low peptide bond specificity of this enzyme [Barrett, A.J. and N. D. Rawlings, Biochem. Soc. Transactions (1991) 19: 707-715].Only a few reports have described proteins of prokaryotic origin thatare resistant to the enzymatic degradation of proteinase K. Proteinase Kresistant proteins have been found in Leptospira species [Nicholson, V.M. and J. F. Prescott, Veterinary Microbiol. (1993) 36:123-138],Mycoplasma species [Butler, G. H. et al. Infec. Immun. (1991)59:1037-1042], Spiroplasma mirum [Bastian, F. O. et al. J. Clin.Microbiol. (1987) 25:2430-2431] and in viruses and prions Onodera, T. etal. Microbiol. Immunol. (1993) 37:311-316; Prusiner, S. B. et al. Proc.Nat. Acad. Sci. USA (1993) 90:2793-2797]. Herein, we describe the use ofthis protein as a means for the improved prevention, treatment anddiagnosis of Neisseria meningitidis infections.

Thus according to one aspect of the invention we provide a highlyconserved, immunologically accessible Neisseria meningitidis surfaceprotein, and fragments, analogues, and derivatives thereof. As usedherein, “Neisseria meningitidis surface protein” means any Neisseriameningitidis surface protein encoded by a naturally occurring Neisseriameningitidis gene. The Neisseria meningitidis protein according to theinvention may be of natural origin, or may be obtained through theapplication of molecular biology with the object of producing arecombinant protein, or fragment thereof.

As used herein, “highly conserved” means that the gene for the Neisseriameningitidis surface protein and the protein itself exist in greaterthan 50% of known strains of Neisseria meningitidis. Preferably, thegene and its protein exist in greater than 99% of known strains ofNeisseria meningitidis. Examples 2 and 4 set forth methods by which oneof skill in the art would be able to test a variety of differentNeisseria meningitidis surface proteins to determine if they are “highlyconserved”.

As used herein, immunologically accessible means that the Neisseriameningitidis surface protein is present at the surface of the organismand is accessible to antibodies. Example 2 sets forth methods by whichone of skill in the art would be able to test a variety of differentNeisseria meningitidis surface proteins to determine if they are“immunologically accessible”. Immunological accessibility may bedetermined by other methods, including an agglutination assay, an ELISA,a RIA, an immunoblotting assay, a dot-enzyme assay, a surfaceaccessibility assay, electron microscopy, or a combination of theseassays.

As used herein, “fragments” of the Neisseria meningitidis surfaceprotein include polypeptides having at least one peptide epitope, oranalogues and derivatives thereof. Peptides of this type may be obtainedthrough the application of molecular biology or synthesized usingconventional liquid or solid phase peptide synthesis techniques.

As used herein, “analogues” of the Neisseria meningitidis surfaceprotein include those proteins, or fragments thereof, wherein one ormore amino acid residues in the naturally occurring sequence is replacedby another amino acid residue, providing that the overall functionalityand protective properties of this protein are preserved. Such analoguesmay be produced synthetically, or by recombinant DNA technology, forexample, by mutagenesis of a naturally occurring Neisseria meningitidissurface protein. Such procedures are well known in the art.

For example, one such analogue is selected from the recombinant proteinthat may be produced from the gene for the 22 kDa protein from Neisseriagonorrhoeae strain b2, as depicted in FIG. 10. A further analog may beobtained from the isolation of the corresponding gene from Neisserialactamica.

As used herein, a “derivative” of the Neisseria meningitidis surfaceprotein is a protein or fragment thereof that has been covalentlymodified, for example, with dinitrophenol, in order to render itimmunogenic in humans. The derivatives of this invention also includederivatives of the amino acid analogues of this invention.

It will be understood that by following the examples of this invention,one of skill in the art may determine without undue experimentationwhether a particular fragment, analogue or derivative would be useful inthe diagnosis, prevention or treatment of Neisseria meningitidisdiseases.

This invention also includes polymeric forms of the Neisseriameningitidis surface proteins, fragments, analogues and derivatives.These polymeric forms include, for example, one or more polypeptidesthat have been crosslinked with crosslinkers such as avidin/biotin,gluteraldehyde or dimethylsuberimidate. Such polymeric forms alsoinclude polypeptides containing two or more tandem or invertedcontiguous Neisseria meningitidis sequences, produced frommulticistronic mRNAs generated by recombinant DNA technology.

This invention provides substantially pure Neisseria meningitidissurface proteins. The term “substantially pure” means that the Neisseriameningitidis surface protein according to the invention is free fromother proteins of Neisseria meningitidis origin. Substantially pureNeisseria meningitidis surface protein preparations can be obtained by avariety of conventional processes, for example the procedure describedin Examples 3 and 11.

In a further aspect, the invention particularly provides a 22 kDasurface protein of Neisseria meningitidis having the amino acid sequenceof FIG. 1 (SEQ ID NO:2), or a fragment, analogue or derivative thereof.

In a further aspect, the invention particularly provides a 22 kDasurface protein of Neisseria meningitidis having the amino acid sequenceof FIG. 8 (SEQ ID NO:4), FIG. 9 (SEQ ID NO:6) or a fragment, analogue orderivative thereof. Such a fragment may be selected from the peptideslisted in FIG. 15 (SEQ ID NO:9 to SEQ ID NO:26).

In a further aspect, the invention provides a 22 kDa surface protein ofNeisseria gonorrhoeae having the amino acid sequence of FIG. 10 (SEQ IDNO:8), or a fragment, analogue or derivative thereof. As will beapparent from the above, any reference to the Neisseria meningitidis 22kDa protein also encompasses 22 kDa proteins isolated from, or made fromgenes isolated from other species of Neisseriacae such as Neisseriagonorrhoeae or Neisseria lactamica.

A Neisseria meningitidis 22 kDa surface protein according to theinvention may be further characterized by one or more of the followingfeatures:

(1) it has an approximate molecular weight of 22 kDa as evaluated onSDS-PAGE gel;

(2) its electrophoretic mobility on SDS-PAGE gel is not modified bytreatment with reducing agents;

(3) it has an isoelectric point (pl) in a range around pl 8 to pl 10;

(4) it is highly resistant to degradation by proteolytic enzymes such asa-chymotrypsin, trypsin and proteinase K;

(5) periodate oxidation does not abolish the specific binding ofantibody directed against the Neisseria meningitidis 22 kDa surfaceprotein;

(6) it is a highly conserved antigen;

(7) it is accessible to antibody at the surface of intact Neisseriameningitidis organisms;

(8) it can induce the production of bactericidal antibodies;

(9) it can induce the production of antibodies that can protect againstexperimental infection;

(10) it can induce, when injected into an animal host, the developmentof an immunological response that can protect against Neisseriameningitidis infection.

This invention also provides, for the first time, a DNA sequence codingfor the Neisseria meningitidis 22 kDa surface protein (SEQ ID NO:1,NO:3, NO:5, and NO:7). The preferred DNA sequences of this invention areselected from the group consisting of:

(a) the DNA sequence of FIG. 1 (SEQ ID NO:1);

(b) the DNA sequence of FIG. 8 (SEQ ID NO:3);

(c) the DNA sequence of FIG. 9 (SEQ ID NO:5);

(d) the DNA sequence of FIG. 10 (SEQ ID NO:7);

(e) analogues or derivatives of the foregoing DNA sequences;

(f) DNA sequences degenerate to any of the foregoing DNA sequences; and

(g) fragments of any of the foregoing DNA sequences;

wherein said sequences encode a product that displays the immunologicalactivity of the Neisseria meningitidis 22 kDa surface protein.

Such fragments are preferably peptides as depicted in FIG. 15 (SEQ IDNO:9, through SEQ ID NO:26).

Preferably, this invention also provides, for the first time, a DNAsequence coding for the Neisseria meningitidis 22 kDa surface protein(SEQ ID NO:1). More preferred DNA sequences of this invention areselected from the group consisting of:

(a) the DNA sequence of FIG. 1 (SEQ ID NO:1);

(b) analogues or derivatives of the foregoing DNA sequences;

(c) DNA sequences degenerate to any of the foregoing DNA sequences; and

(d) fragments of any of the foregoing DNA sequences; wherein saidsequences encode a product that displays the immunological activity ofthe Neisseria meningitidis 22 kDa surface protein.

Analogues and derivatives of the Neisseria meningitidis 22 kDa surfaceprotein coding gene will hybridize to the 22 kDa surface protein-codinggene under the conditions described in Example 4.

For purposes of this invention, the fragments, analogues and derivativesof the Neisseria meningitidis 22 kDa surface protein have the“immunological activity” of the Neisseria meningitidis 22 kDa surfaceprotein if they can induce, when injected into an animal host, thedevelopment of an immunological response that can protect againstNeisseria meningitidis infection. One of skill in the art may determinewhether a particular DNA sequence encodes a product that displays theimmunological activity of the Neisseria meningitidis 22 kDa surfaceprotein by following the procedures set forth herein in Example 6.

The Neisseria meningitidis surface proteins of this invention may beisolated by a method comprising the following steps:

a) isolating a culture of Neisseria meningitidis bacteria,

b) isolating an outer membrane portion from the culture of the bacteria;and

c) isolating said antigen from the outer membrane portion.

In particular, the foregoing step (c) may include the additional stepsof treating the Neisseria meningitidis outer membrane protein extractswith proteinase K, followed by protein fractionation using conventionalseparation techniques such as ion exchange and gel chromatography andelectrophoresis.

Alternatively and preferably, the Neisseria meningitidis surfaceproteins of this invention may be produced by the use of molecularbiology techniques, as more particularly described in Example 3 herein.The use of molecular biology techniques is particularly well-suited forthe preparation of substantially pure recombinant Neisseria meningitidis22 kDa surface protein.

Thus according to a further aspect of the invention we provide a processfor the production of recombinant Neisseria meningitidis 22 kDa surfaceprotein, including fragments, analogues and derivatives thereof,comprising the steps of (1) culturing a unicellular host organismtransformed with a recombinant DNA molecule including a DNA sequencecoding for said protein, fragment, analogue or derivative and one ormore expression control sequences operatively linked to the DNAsequence, and (2) recovering a substantially pure protein, fragment,analogue or derivative.

As is well known in the art, in order to obtain high expression levelsof a transfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host. Preferably, the expressioncontrol sequences, and the gene of interest, will be contained in anexpression vector that further comprises a bacterial selection markerand origin of replication. If the expression host is a eukaryotic cell,the expression vector should further comprise an expression markeruseful in the expression host.

A wide variety of expression host/vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors for eukaryotic hosts include, for example, vectors comprisingexpression control sequences from SV40, bovine papilloma virus,adenovirus and cytomegalovirus. Useful expression vectors for bacterialhosts include known bacterial plasmids, such as plasmids from E. coli,including col E1, pCR1, pBR322, pMB9 and their derivatives, wider hostrange plasmids, such as RP4, phage DNAs, e.g., the numerous derivativesof phage lambda, e.g., NM989, and other DNA phages, such as M13 andfilamentous single stranded DNA phages. Useful expression vectors foryeast cells include the 2 mu plasmid and derivatives thereof. Usefulvectors for insect cells include pVL 941.

In addition, any of a wide variety of expression control sequences maybe used in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include the expression controlsequences associated with structural genes of the foregoing expressionvectors. Examples of useful expression control sequences include, forexample, the early and late promoters of SV40 or adenovirus, the lacsystem, the trp system, the TAC or TRC system, the major operator andpromoter regions of phage lambda, the control regions of fd coatprotein, the promoter for 3-phosphoglycerate kinase or other glycolyticenzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters ofthe yeast alpha-mating system and other sequences known to controlexpression of genes of prokaryotic and eukaryotic cells or theirviruses, and various combinations thereof. The Neisseria meningitidis 22kDa surface protein's expression control sequence is particularly usefulin the expression of the Neisseria meningitidis 22 kDa surface proteinin E. Coli (Example 3).

Host cells transformed with the foregoing vectors form a further aspectof this invention. A wide variety of unicellular host cells are usefulin expressing the DNA sequences of this invention. These hosts mayinclude well known eukaryotic and prokaryotic hosts, such as strains ofE. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cellssuch as Spodoptera frugiperda (SF9), animal cells such as CHO and mousecells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40,and BMT 10, and human cells and plant cells in tissue culture. Preferredhost organisms include bacteria such as E. Coli and Bacillus subtilisand mammalian cells in tissue culture.

It should of course be understood that not all vectors and expressioncontrol sequences will function equally well to express the DNAsequences of this invention. Neither will all hosts function equallywell with the same expression system. However, one of skill in the artmay make a selection among these vectors, expression control sequencesand hosts without undue experimentation and without departing from thescope of this invention. For example, in selecting a vector, the hostmust be considered because the vector must replicate in it. The vectorscopy number, the ability to control that copy number, and the expressionof any other proteins encoded by the vector, such as antibiotic markers,should also be considered.

In selecting an expression control sequence, a variety of factors shouldalso be considered. These include, for example, the relative strength ofthe sequence, its controllability, and its compatibility with the DNAsequences of this invention, particularly as regards potential secondarystructures. Unicellular hosts should be selected by consideration oftheir compatibility with the chosen vector, the toxicity of the productcoded for by the DNA sequences of this invention, their secretioncharacteristics, their ability to fold the protein correctly, theirfermentation or culture requirements, and the ease of purification fromthem of the products coded for by the DNA sequences of this invention.

Within these parameters, one of skill in the art may select variousvector/expression control sequence/host combinations that will expressthe DNA sequences of this invention on fermentation or in large scaleanimal culture.

The polypeptides encoded by the DNA sequences of this invention may beisolated from the fermentation or cell culture and purified using any ofa variety of conventional methods. One of skill in the art may selectthe most appropriate isolation and purification techniques withoutdeparting from the scope of this invention.

The Neisseria meningitidis surface proteins of this invention are usefulin prophylactic, therapeutic and diagnostic compositions for preventing,treating and diagnosing diseases caused by Neisseria meningitidisinfection.

The Neisseria meningitidis surface proteins of this invention are usefulin prophylactic, therapeutic and diagnostic compositions for preventing,treating and diagnosing diseases caused by Neisseria gonorrhoeae, orNeisseria lactamica infection.

The Neisseria meningitidis surface proteins according to this inventionare particularly well-suited for the generation of antibodies and forthe development of a protective response against Neisseria meningitidisdiseases.

The Neisseria meningitidis surface proteins according to this inventionare particularly well-suited for the generation of antibodies and forthe development of a protective response against Neisseria gonorrhoeaeor Neisseria lactamica diseases.

In particular, we provide a Neisseria meningitidis 22 kDa surfaceprotein having an amino acid sequence of FIG. 1 (SEQ ID NO:2) or afragment, analogue, or derivative thereof for use as an immunogen and asa vaccine.

In particular, we provide a Neisseria meningitidis 22 kDa surfaceprotein having an amino acid sequence of FIG. 1 (SEQ ID NO:2), FIG. 8(SEQ ID NO:4), FIG. 9 (SEQ ID NO:6), or FIG. 10 (SEQ ID NO:8), or afragment, analogue, or derivative thereof for use as an immunogen and asa vaccine.

Standard immunological techniques may be employed with the Neisseriameningitidis surface proteins in order to use them as immunogens and asvaccines. In particular, any suitable host may be injected with apharmaceutically effective amount of the Neisseria meningitidis 22 kDasurface protein to generate monoclonal or polyvalent anti-Neisseriameningitidis antibodies or to induce the development of a protectiveimmunological response against Neisseria meningitidis diseases. Prior toinjection of the host, the Neisseria meningitidis surface proteins maybe formulated in a suitable vehicle, and thus we provide apharmaceutical composition comprising one or more Neisseria meningitidissurface antigens or fragments thereof. Preferably, the antigen is theNeisseria meningitidis 22 kDa surface protein or fragments, analogues orderivatives thereof together with one or more pharmaceuticallyacceptable excipients. As used herein, “pharmaceutically effectiveamount” refers to an amount of one or more Neisseria meningitidissurface antigens or fragments thereof that elicits a sufficient titer ofanti-Neisseria meningitidis antibodies to treat or prevent Neisseriameningitidis infection.

The Neisseria meningitidis surface proteins of this invention may alsoform the basis of a diagnostic test for Neisseria meningitidisinfection. Several diagnostic methods are possible. For example, thisinvention provides a method for the detection of Neisseria meningitidisantigen in a biological sample containing or suspected of containingNeisseria meningitidis antigen comprising:

a) isolating the biological sample from a patient;

b) incubating an anti-Neisseria meningitidis 22 kDa surface proteinantibody or fragment thereof with the biological sample to form amixture; and

c) detecting specifically bound antibody or bound fragment in themixture which indicates the presence of Neisseria meningitidis antigen.

Preferred antibodies in the foregoing diagnostic method are Me-i andMe-7.

Alternatively, this invention provides a method for the detection ofantibody specific to Neisseria meningitidis antigen in a biologicalsample containing or suspected of containing said antibody comprising:

a) isolating the biological sample from a patient;

b) incubating a Neisseria meningitidis surface protein of this inventionor fragment thereof with the biological sample to form a mixture; and

c) detecting specifically bound antigen or bound fragment in the mixturewhich indicates the presence of antibody specific to Neisseriameningitidis antigen. One of skill in the art will recognize that thisdiagnostic test may take several forms, including an immunological testsuch as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassayor a latex agglutination assay, essentially to determine whetherantibodies specific for the protein are present in an organism.

The DNA sequences of this invention may also be used to design DNAprobes for use in detecting the presence of the pathogenic Neisseriabacteria in a biological suspected of containing such bacteria. Thedetection method of this invention comprises the steps of:

a) isolating the biological sample from a patient;

b) incubating a DNA probe having a DNA sequence of this invention withthe biological sample to form a mixture; and

c) detecting specifically bound DNA probe in the mixture which indicatesthe presence of Neisseria bacteria.

Preferred DNA probes have the base pair sequence of FIG. 1 (SEQ IDNO:1), FIG. 8 (SEQ ID NO:3), FIG. 9 (SEQ ID NO:5), or FIG. 10 (SEQ IDNO:7), or consensus sequence of FIG. 11 (SEQ ID NO:9).

A more preferred DNA probe has the 525 base pair sequence of FIG. 1 (SEQID NO:1).

The DNA probes of this invention may also be used for detectingcirculating Neisseria meningitidis nucleic acids in a sample, forexample using a polymerase chain reaction, as a method of diagnosingNeisseria meningitidis infections. The probe may be synthesized usingconventional techniques and may be immobilized on a solid phase, or maybe labeled with a detectable label.

A preferred DNA probe for this application is an oligomer having asequence complementary to at least about 6 contiguous nucleotides of theNeisseria meningitidis 22 kDa surface protein gene of FIG. 1 (SEQ IDNO:1), FIG. 8 (SEQ ID NO:3), FIG. 9 (SEQ ID NO:5), FIG. 10 (SEQ IDNO:7), or consensus sequence of FIG. 11 (SEQ ID NO:9).

A more preferred DNA probe for this application is an oligomer having asequence complementary to at least about 6 contiguous nucleotides of theNeisseria meningitidis 22 kDa surface protein gene of FIG. 1 (SEQ IDNO:1).

Another diagnostic method for the detection of Neisseria meningitidis ina patient comprises the steps of:

a) labeling an antibody of this invention or fragment thereof with adetectable label;

b) administering the labeled antibody or labeled fragment to thepatient; and

c) detecting specifically bound labeled antibody or labeled fragment inthe patient which indicates the presence of Neisseria meningitidis.

For purification of any anti-Neisseria meningitidis surface proteinantibody, use may be made of affinity chromatography employing animmobilized Neisseria meningitidis surface protein as the affinitymedium.

Thus according to another aspect of the invention we provide a Neisseriameningitidis 22 kDa surface protein having an amino acid sequence whichincludes the sequence of FIG. 1 (SEQ ID NO:2), FIG. 8 (SEQ ID NO:4),FIG. 9 (SEQ ID NO:6), or FIG. 10 (SEQ ID NO;8), or portion thereof or ananalogue thereof, covalently bound to an insoluble support.

Thus according to a preferred aspect of the invention we provide aNeisseria meningitidis 22 kDa surface protein having an amino acidsequence which includes the sequence of FIG. 1 (SEQ ID NO:2), or portionthereof or an analogue thereof, covalently bound to an insolublesupport.

A further feature of the invention is the use of the Neisseriameningitidis surface proteins of this invention as immunogens for theproduction of specific antibodies for the diagnosis and in particularthe treatment of Neisseria meningitidis infection. Suitable antibodiesmay be determined using appropriate screening methods, for example bymeasuring the ability of a particular antibody to passively protectagainst Neisseria meningitidis infection in a test model. One example ofan animal model is the mouse model described in the Examples herein. Theantibody may be a whole antibody or an antigen-binding fragment thereofand may in general belong to any immunoglobulin class. The antibody orfragment may be of animal origin, specifically of mammalian origin andmore specifically of murine, rat or human origin. It may be a naturalantibody or a fragment thereof, or if desired, a recombinant antibody orantibody fragment. The term recombinant antibody or antibody fragmentmeans antibody or antibody fragment which were produced using molecularbiology techniques. The antibody or antibody fragments may be ofpolyclonal, or preferentially, monoclonal origin. It may be specific fora number of epitopes associated with the Neisseria meningitidis surfaceproteins but it is preferably specific for one. Preferably, the antibodyor fragments thereof will be specific for one or more epitopesassociated with the Neisseria meningitidis 22 kDa surface protein. Alsopreferred are the monoclonal antibodies Me-1 and Me-7 described herein.

EXAMPLES

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any manner.

Example 1 describes the treatment of Neisseria meningitidis outermembrane preparation with proteolytic enzymes and the subsequentidentification of the Neisseria meningitidis 22 kDa surface protein.

Example 2 describes the preparation of monoclonal antibodies specificfor Neisseria meningitidis 22 kDa surface protein.

Example 3 describes the preparation of Neisseria meningitidisrecombinant 22 kDa surface protein.

Example 4 describes the use of DNA probes for the identification oforganisms expressing the Neisseria meningitidis 22 kDa surface protein.

Example 5 describes the use of an anti-Neisseria meningitidis 22 kDasurface protein monoclonal antibody to protect mice against Neisseriameningitidis infection.

Example 6 describes the use of purified recombinant 22 kDa surfaceprotein to induce a protective response against Neisseria meningitidisinfection.

Example 7 describes the identification of the sequence for the 22 kDaprotein and protein-coding gene for other strains of Neisseriameningitidis (MCH88, and Z4063), and one strain of Neisseriagonorrhoeae.

Example 8 describes the immunological response of rabbits and monkeys tothe 22 kDa Neisseria meningitidis surface protein.

Example 9 describes the procedure used to map the differentimmunological epitopes of the 22 kDa Neisseria meningitidis surfaceprotein.

Example 10 describes the induction by heat of an expression vector forthe large scale production of the 22 kDa surface protein.

Example 11 describes a purification process for the 22 kDa surfaceprotein when produced by recombinant technology.

Example 12 describes the use of 22 kDa surface protein as a humanvaccine.

Example 1 Treatment of Neisseria Meningitidis Outer MembranePreparations with Proteolytic Enzymes and the Subsequent Identificationof an Enzyme Resistant Neisseria Meningitidis 22 kDa Surface Protein

Several antigenic preparations derived from whole cell, lithiumchloride, or sarcosyl extracts were used to study the ultrastructure ofNeisseria meningitidis outer membrane. The outer membrane ofGram-negative bacteria acts as an interface between the environment andthe interior of the cell and contains most of the antigens that arefreely exposed to the host immune defense. The main goal of the studywas the identification of new antigens which can induce a protectiveresponse against Neisseria meningitidis. One approach used by theinventors to identify such antigens, was the partial disruption of theantigenic preparations mentioned above with proteolytic enzymes. Theantigenic determinants generated by the enzymatic treatments could thenbe identified by the subsequent analysis of the immunological andprotective properties of these treated antigenic preparations. To oursurprise we observed after electrophoretic resolution of Neisseriameningitidis lithium chloride outer membrane extracts, that one lowmolecular weight band, which was stained with Coomassie Brilliant BlueR-250, was not destroyed by proteolytic enzyme treatments. CoomassieBlue is used to stain proteins and peptides and has no or very littleaffinity for the polysaccharides or lipids which are also key componentsof the outer membrane. The fact that this low molecular weight antigenwas stained by Coomassie blue suggested that at least part of it is madeup of polypeptides that are not digested by proteolytic enzymes, or thatare protected against the action of the enzymes by other surfacestructures. Moreover, as demonstrated below the very potent enzymeproteinase K did not digest this low molecular weight antigen even afterextensive treatments.

Lithium chloride extraction was used to obtain the outer membranepreparations from different strains of Neisseria meningitidis and wasperformed in a manner previously described by the inventors [Brodeur etal., Infect. Immun., 50, p. 510 (1985)]. The protein content of thesepreparations were determined by the Lowry method adapted to membranefractions [Lowry et al., J. Biol. Chem. 193, p. 265 (1951)]. Outermembrane preparations derived from Neisseria meningitidis strain 608B(B:2a:P1.2) were treated for 24 hours at 37° C. and continuous shakingwith either α-chymotrypsin from bovine pancreas (E.C. 3.4.21.1) (Sigma)or trypsin type 1 from bovine pancreas (E.C. 3.4.21.4) (Sigma). Theenzyme concentration was adjusted at 2 mg per mg of protein to betreated. The same outer membrane preparations were also treated withdifferent concentrations (0.5 to 24 mg per mg of protein) of ProteinaseK from Tritirachium album limber (Sigma or Boehringer Mannheim, Laval,Canada) (E.C. 3.4.21.14). In order to promote protein digestion byproteinase K, different experimental conditions were used. The sampleswere incubated for 1 hour, 2 hours, 24 hours or 48 hours at 37° C. or56° C. with or without shaking. The pH of the mixture samples wasadjusted at either pH 7.2 or pH 9.0. One % (vol/vol) of sodium dodecylsulfate (SDS) was also added to certain samples. Immediately aftertreatment the samples were resolved by SDS-PAGE gel electrophoresisusing the MiniProteanII® (Bio-Rad, Mississauga, Ontario, Canada) systemon 14% (wt/vol) gels according to the manufacturer's instructions.Proteins were heated to 100° C. for 5 minutes with 2-mercaptoethanol andSDS, separated on 14% SDS gels, and stained with Coomassie BrilliantBlue R-250.

FIG. 2 presents the migration profile on 14% SDS-PAGE gel of theproteins present in outer membrane preparations derived from Neisseriameningitidis strain 608B (B:2a:P1.2) after treatment at 37° C. for 24hours with α-chymotrypsin and trypsin. Extensive proteolytic digestionof the high molecular weight proteins and of several major outermembrane proteins can be observed for the treated samples (FIG. 2, lanes3 and 4) compared to the untreated control (FIG. 2, lane 2). On thecontrary, a protein band with an apparent molecular weight of 22 kDa wasnot affected even after 24 hours of contact with either proteolyticenzyme.

This unique protein was further studied using a more aggressiveproteolytic treatment with Proteinase K (FIG. 3). Proteinase K is one ofthe most powerful proteolytic enzymes since it has a low peptide bondspecificity and wide pH optimum. Surprisingly, the 22 kDa protein wasresistant to digestion by 2 International Units (IU) of proteinase K for24 hours at 56° C. (FIG. 3, lane 2). This treatment is often used in ourlaboratory to produce lipopolysaccharides or DNA that are almost free ofproteins. Indeed, only small polypeptides can be seen after such anaggressive proteolytic treatment of the outer membrane preparation.Furthermore, longer treatments, up to 48 hours, or higher enzymeconcentrations (up to 24 IU) did not alter the amount of the 22 kDaprotein. The amount and migration on SDS-PAGE gel of the 22 kDa proteinwere not affected when the pH of the reaction mixtures was increased topH 9.0, or when 1.0% of SDS, a strong protein denaturant was added (FIG.3, lanes 4, 6 and 8). The combined use of these two denaturingconditions would normally result in the complete digestion of theproteins present in the outer membrane preparations, leaving only aminoacid residues. Polypeptides of low molecular weight were often observedin the digests and were assumed to be fragments of sensitive proteinsnot effectively digested during the enzymatic treatments. Thesefragments were most probably protected from further degradation by thecarbohydrates and lipids present in the outer membrane. The bands withapparent molecular weight of 28 kDa and 34 kDa which are present intreated samples are respectively the residual enzyme and a contaminatingprotein present in all enzyme preparations tested.

Interestingly, this study about the resistance of the 22 kDa protein toproteases indicated that another protein band with apparent molecularweight of 18 kDa seems to be also resistant to enzymatic degradation(FIG. 3 a). Clues about this 18 kDa protein band were obtained when themigration profiles on SDS-PAGE gels of affinity purified recombinant 22kDa protein were analyzed (FIG. 3 b). The 18 kDa band was apparent onlywhen the affinity purified recombinant 22 kDa protein was heated for anextended period of time in sample buffer containing the detergent SDSbefore it was applied on the gel. N-terminal amino acid analysis usingthe Edman degradation (Example 3) clearly established that the aminoacid residues (E-G-A-S-G-F-Y-V-Q) (SEQ ID NO: 31) identified on the 18kDa band corresponded to the amino acids 1-9 (SEQ ID NO:1). Theseresults indicate that the 18 and 22 kDa bands as seen on the SDS-PAGE isin fact derived from the same protein. This last result also indicatesthat the leader sequence is cleaved from the mature 18 kDa protein.Further studies will be done to identify the molecular modificationsexplaining this shift in apparent molecular weight and to evaluate theirimpact on the antigenic and protective properties of the protein.

In conclusion, the discovery of a Neisseria meningitidis outer membraneprotein with the very rare property of being resistant to proteolyticdigestion warranted further study of its molecular and immunologicalcharacteristics. The purified recombinant 22 kDa surface proteinproduced by Escherichia coli in Example 3 is also highly resistant toproteinase K digestion. We are presently trying to understand themechanism which confers to the Neisseria meningitidis 22 kDa surfaceprotein this unusual resistance to proteolytic enzymes.

Example 2 Generation of Monoclonal Antibodies Specific for the 22 kDaNeisseria Meningitidis Surface Protein

The monoclonal antibodies described herein were obtained from threeindependent fusion experiments. Female Balb/c mice (Charles RiverLaboratories, St-Constant, Quebec, Canada) were immunized with outermembrane preparations obtained from Neisseria meningitidis strains 604A,608B and 2241C respectively serogrouped A, B and C. The lithium chlorideextraction used to obtain these outer membrane preparations wasperformed in a manner previously described by the inventors. [Brodeur etal., Infect. Immun. 50, p. 510 (1985)]. The protein content of thesepreparations were determined by the Lowry method adapted to membranefractions [Lowry et al., J. Biol. Chem. 193, p. 265 (1951)]. Groups ofmice were injected intraperitoneally or subcutaneously twice, atthree-week intervals with 10 mg of different combinations of the outermembrane preparations described above. Depending on the group of mice,the adjuvants used for the immunizations were either Freund's completeor incomplete adjuvant (Gibco Laboratories, Grand Island, N.Y.), orQuilA (CedarLane Laboratories, Hornby, Ont., Canada). Three days beforethe fusion procedure, the immunized mice received a final intravenousinjection of 10 mg of one of the outer membrane preparations describedabove. The fusion protocol used to produce the hybridoma cell linessecreting the desired monoclonal antibody was described previously bythe inventors [Hamel et al., J. Med. Microbiol., 25, p. 2434 (1987)].The class, subclass and light-chain type of monoclonal antibodies Me-1,Me-2, Me-3, Me-5, Me-6 and Me-7 were determined by ELISA as previouslyreported [Martin et al., J. Clin. Microbiol., 28, p. 1720 (1990)] andare presented in Table 1.

The specificity of the monoclonal antibodies was established usingWestern immmoblotting following the method previously described by theinventors [Martin et al., Eur. J. Immunol. 18, p. 601 (1988)] with thefollowing modifications. Outer membrane preparations obtained fromdifferent strains of Neisseria meningitidis were resolved on 14%SDS-PAGE gels. The proteins were transferred from the gels tonitrocellulose membranes using a semi-dry apparatus (Bio-Rad). A currentof 60 mA per gel (6×10 cm) was applied for 10 minutes in the electroblotbuffer consisting of 25 mM Tris-HCl, 192 mM glycine and 20% (vol/vol)methanol, pH 8.3. The Western immunoblotting experiments clearlyindicated that the monoclonal antibodies Me-1, Me-2, Me-3, Me-5, Me-6and Me-7 recognized their specific epitopes on the Neisseriameningitidis 22 kDa protein (FIG. 4A). Analysis of the SDS-PAGE gels andthe corresponding Western immunoblots also indicated that the apparentmolecular weight of this protein does not vary from one strain toanother. However, the amount of protein present in the outer membranepreparations varied from one strain to another and was not related tothe serogroup of the strain. Moreover, these monoclonal antibodies stillrecognized their epitopes on the Neisseria meningitidis 22 kDa surfaceprotein after treatment of the outer membrane preparation with 2 IU ofproteinase K per mg of protein (treatment described in Example 1, supra)(FIG. 4B). Interestingly, the epitopes remained intact after the enzymedigestion thus confirming that even if they are accessible in themembrane preparation to the antibodies they are not destroyed by theenzyme treatment. This latter result suggested that the mechanism whichexplains the observed proteinase K resistance is most probably notrelated to surface structures blocking the access of the enzyme to theprotein, or to the protection offered by the membrane to proteins whichare deeply embedded. While not shown in FIG. 4, the results of theimmunoblots for Me-1 were consistent with the results for the other fivemonoclonal antibodies.

A series of experiments were performed to partially characterize theNeisseria meningitidis 22 kDa surface protein and to differentiate itfrom the other known meningococcal surface proteins. No shift inapparent molecular weight on SDS-PAGE gel of the Neisseria meningitidis22 kDa surface protein was noted when outer membrane preparations wereheated at 100° C. for 5 minutes, or at 37° C. and 56° C. for 30 minutesin electrophoresis sample buffer with or without 2-mercaptoethanol. Thisindicated that the migration of the 22 kDa surface protein, when presentin the outer membrane, was not heat- or 2-mercaptoethanol-modifiable.

Sodium periodate oxidation was used to determine if the monoclonalantibodies reacted with carbohydrate epitopes present in the outermembrane preparations extracted from Neisseria meningitidis organisms.The method used to perform this experiment was previously described bythe inventors. [Martin et al., Infect. Immun., 60, pp. 2718-2725(1992)]. Treatment of outer membrane preparations with 100 mM of sodiumperiodate for 1 hour at room temperature did not alter the reactivity ofthe monoclonal antibodies toward the Neisseria meningitidis 22 kDAsurface protein. This treatment normally abolishes the binding ofantibodies that are specific for carbohydrates.

Monoclonal antibody 2-1-CA2 (provided by Dr. A. Bhattacharjee. WalterReed Army Institute of Research, Washington, D.C.) is specific for thelip protein (also called H.8), a surface antigen common to allpathogenic Neisseria species. The reactivity of this monoclonal antibodywith outer membrane preparations was compared to the reactivity ofmonoclonal antibody Me-5. The lip-specific monoclonal antibody reactedwith a protein band having an apparent molecular weight of 30 kDa, whilemonoclonal antibody Me-5 reacted with the protein band of 22 kDa. Thisresult clearly indicates that there is no relationship between Neisseriameningitidis 22 kDa surface protein and the lip protein, another highlyconserved outer membrane protein.

To verify the exposure of the 22 kDa protein at the surface of intactNeisseria meningitidis bacterial cells, a radioimmunoassay was performedas previously described by the inventors [Proulx et al., Infec. Immun.,59, p. 963 (1991)]. Six-hour and 18-hour bacterial cultures were usedfor this assay. The six monoclonal antibodies were reacted with 9Neisseria meningitidis strains (the serogroup of the strain is indicatedin parentheses on FIG. 5), 2 Neisseria gonorrhoeae strains (“NG”), 2Moraxella catarrhalis strains (“MC”) and 2 Neisseria lactamica strains(“NL”). The radioimmunoassay confirmed that the epitopes recognized bythe monoclonal antibodies are exposed at the surface of intact Neisseriameningitidis isolates of different serotypes and serogroups and shouldalso be accessible to the proteolytic enzymes (FIG. 5). The monoclonalantibodies bound strongly to their target epitopes on the surface of allNeisseria meningitidis strains tested. The recorded binding values(between 3,000 to 35,000 CPM), varied from one strain to another, andwith the physiological state of the bacteria. A Haemophilus influenzaeporin-specific monoclonal antibody was used as a negative control foreach bacterial strain. Counts below 500 CPM were obtained andsubsequently subtracted from each binding value. With respect to theNeisseria meningitidis strains tested in this assay, the results shownin FIG. 5 for monoclonal antibodies Me-5 and Me-7 are representative ofthe results obtained with monoclonal antibodies Me-1, Me-2, Me-3 andMe-6. With respect to the other bacterial strains tested, the bindingactivities shown for Me-7 are representative of the binding activitiesobtained with other monoclonal antibodies that recognized the samebacterial strain.

The antigenic conservation of the epitopes recognized by the monoclonalantibodies was also evaluated. A dot enzyme immunoassay was used for therapid screening of the monoclonal antibodies against a large number ofbacterial strains. This assay was performed as previously described bythe inventors [Lussier et al., J. Immunoassay, 10, p. 373 (1989)]. Acollection of 71 Neisseria meningitidis strains was used in this study.The sample included 19 isolates of serogroup A, 23 isolates of serogroupB, 13 isolates of serogroup C, 1 isolate of serogroup 29E, 6 isolates ofserogroup W-135, 1 isolate of serogroup X, 2 isolates of serogroup Y, 2isolates of serogroup Z, and 4 isolates that were not serogrouped(“NS”). These isolates were obtained from the Caribbean EpidemiologyCentre, Port of Spain, Trinidad; Children's Hospital of Eastern Ontario,Ottawa, Canada; Department of Saskatchewan Health, Regina, Canada;Laboratoire de Sante Publique du Quebec, Montreal, Canada; Max-PlanckInstitut fur Molekulare Genetik, Berlin, FRG; Montreal ChildrenHospital, Montreal, Canada; Victoria General Hospital, Halifax, Canada;and our own strains collection. The following bacterial species werealso tested: 16 Neisseria gonorrhoeae, 4 Neisseria cinerea, 5 Neisserialactamica, 1 Neisseria flava, 1 Neisseria flavescens, 3 Neisseriamucosa, 4 Neisseria perflava/sicca, 4 Neisseria perflava, 1 Neisseriasicca, 1 Neisseria subflava and 5 Moraxella catarrhalis, 1 Alcaligenesfeacalis (ATCC 8750), 1 Citrobacter freundii (ATCC 2080), 1 Edwarsiellatarda (ATCC 15947), 1 Enterobacter cloaca (ATCC 23355), 1 Enterobacteraerogenes (ATCC 13048), 1 Escherichia coli, 1 Flavobacterium odoratum, 1Haemophilus influenzae type b (Eagan strain), 1 Klebsiella pneumoniae(ATCC 13883), 1 Proteus rettgeri (ATCC 25932), 1 Proteus vulgaris (ATCC13315), 1 Pseudomonas aeruginosa (ATCC 9027), 1 Salmonella typhimurium(ATCC 14028), 1 Serrati marcescens (ATCC 8100), 1 Shigella flexneri(ATCC 12022), 1 Shigella sonnei (ATCC 9290). They were obtained from theAmerican Type Culture Collection or a collection held in the LaboratoryCentre for Disease Control, Ottawa, Canada. The reactivities of themonoclonal antibodies with the most relevant Neisseria strains arepresented in Table 1. One monoclonal antibody, Me-7, recognized itsspecific epitope on 100% of the 71 Neisseria meningitidis strainstested. This monoclonal antibody, as well as Me-2, Me-3, Me-5 and Me-6also reacted with certain strains belonging to other Neisserial speciesindicating that their specific epitope is also expressed by otherclosely related Neisseriaceae. Except for a faint reaction with oneNeisseria lactamica strain, monoclonal antibody Me-1 reacted only withNeisseria meningitidis isolates. Me-1 was further tested with anothersample of 177 Neisseria meningitidis isolates and was able to correctlyidentify more than 99% of the total Neisseria meningitidis strainstested. Besides the Neisseria strains presented in Table 1, themonoclonal antibodies did not react with any of the other bacterialspecies mentioned above.

In conclusion, six monoclonal antibodies which specifically reacted withthe Neisseria meningitidis 22 kDa surface protein were generated by theinventors. Using these monoclonal antibodies we demonstrated that theirspecific epitopes are 1) located on a proteinase K resistant 22 kDaprotein present in the outer membrane of Neisseria meningitidis, 2)conserved among Neisseria meningitidis isolates, 3) exposed at thesurface of intact Neisseria meningitidis cells and accessible toantibody, and 4) the reactivity of these monoclonal antibodies with theNeisseria meningitidis 22 kDa surface protein is not modified by atreatment with sodium periodate, suggesting that their specific epitopesare not located on carbohydrates.

Although we found that the migration of the Neisseria meningitidis 22kDa protein is moved to an apparent molecular weight of about 18 kDawhen heated under stringent conditions, we observed that the migrationis not modified by 2-mercaptoethanol treatment.

We also demonstrated that the Neisseria meningitidis 22 kDa surfaceprotein has no antigenic similarity with the lip protein, another lowmolecular weight and highly conserved protein present in the outermembrane of Neisseria meningitidis.

As will be presented in Example 3, these monoclonal antibodies alsoreacted with the purified, recombinant 22 kDa surface protein producedafter transformation of Escherichia coli strain BL21 (DE3) with aplasmid vector pNP2202 containing the gene coding for the Neisseriameningitidis 22 kDa surface protein.

TABLE 1 Reactivity of the monoclonal antibodies with Neisseria isolatesNumber of Neisseria isolates recognized by the monoclonal antibodiesSerogroup of Neisseria meningitidis Moraxella Neisseria Neisseria Iso- AB C 29% W135 X Y Z NS¹ Total catarrhalis gonorrhoeae lactamica Name type(19) (23) (13) (1) (5) (1) (2) (2) (4) (71) (5) (16) (5) Me-1 IgG2a 1922 13 1 6 1 2 2 3 69 0 0 1 (k) Me-2 IgG2a 19 20 13 1 6 0 2 2 4 67 0 2 0(k) Me-3 IgG3 19 22 13 1 6 1 2 2 3 69 0 2 4 (k) Me-5 IgG2a 19 22 13 1 61 2 2 3 69 0 2 0 (k) Me-6 IgG1 19 23 13 1 6 1 2 2 3 70 0 2 4 (k) Me-7IgG2a 19 23 13 1 6 1 2 2 4 71 5 2 4 (k)

Example 3 Molecular Cloning, Sequencing of the Gene, High YieldExpression and Purification of the Neisseria Meningitidis 22 kDa SurfaceProtein A. Molecular Cloning

A LambdaGEM-11 genomic DNA library from Neisseria meningitidis strain608B (B:2a:P1.2) was constructed according to the manufacturer'srecommendations (Promega CO, Madison, Wis.). Briefly, the genomic DNA ofthe 608B strain was partially digested with Sau 3AI, and fragmentsranging between 9 and 23 Kb were purified on agarose gel before beingligated to the Bam HI sites of the LambdaGEM-11 arms. The resultingrecombinant phages were used to infect Escherichia coli strain LE392(Promega) which was then plated onto LB agar plates. Nineteen positiveplaques were identified after the immuno-screening of the library withthe Neisseria meningitidis 22 kDa surface protein-specific monoclonalantibodies of Example 2 using the following protocol. The plates wereincubated 15 minutes at −20° C. to harden the top agar. Nitrocellulosefilters were gently applied onto the surface of the plates for 30minutes at 4° C. to absorb the proteins produced by the recombinantviral clones. The filters were then washed in PBS-Tween 0.02% (vol/vol)and immunoblotted as described previously [Lussier et al., J.Immunoassay, 10, p. 373 (1989)]. After amplification and DNApurification, one viral clone, designated clone 8, which had a 13 Kbinsert was selected for the subcloning experiments. After digestion ofthis clone with Sac I, two fragments of 5 and 8 Kb were obtained. Thesefragments were purified on agarose gel and ligated into the Sac Irestriction site of the low copy number plasmid pWKS30 [Wang andKushner, Gene, 100, p. 195 (1991)]. The recombinant plasmids were usedto transform Escherichia coli strain JM109 (Promega) by electroporation(Bio-Rad, Mississauga, Ont., Canada) following the manufacturer'srecommendations, and the resulting colonies were screened with theNeisseria meningitidis 22 kDa surface protein-specific monoclonalantibodies of Example 2. Positive colonies were observed only when thebacteria were transformed with the plasmid carrying the 8 Kb insert.Western blot analysis (the methodology was described in Example 2) ofthe positive clones showed that the protein expressed by Escherichiacoli was complete and migrated on SDS-PAGE gel like the Neisseriameningitidis 22 kDa surface protein. To further reduce the size of theinsert, a clone containing the 8 Kb fragment was digested with Cla I anda 2.75 Kb fragment was then ligated into the Cla I site of the pWKS30plasmid. Western blot analysis of the resulting clones clearly indicatedonce again that the protein expressed by Escherichia coli was completeand migrated on SDS-PAGE gel like the native Neisseria meningitidis 22kDa surface protein.

After restriction analysis, two clones, designated pNP2202 and pNP2203,were shown to carry the 2.75 Kb insert in opposite orientations and wereselected to proceed with the sequencing of the gene coding for theNeisseria meningitidis 22 kDa surface protein. The “Double StrandedNested Deletion Kit” from Pharmacia Biotech Inc. (Piscataway, N.J.) wasused according to the manufacturer's instructions to generate a seriesof nested deletions from both clones. The resulting truncated insertswere then sequenced from the M13 forward primer present on the pWKS30vector with the “Taq Dye Deoxy Terminator Cycle Sequencing Kit” using anApplied Biosystems Inc. (Foster City, Calif.) automated sequencer model373A according to the manufacturer's recommendations.

B. Sequence Analysis

After the insert was sequenced in both directions, the nucleotidesequence revealed an open reading frame consisting of 525 nucleotides(including the stop codon) encoding a protein composed of 174 amino acidresidues having a predicted molecular weight of 18,000 Daltons and a plof 9.93. The nucleotide and deduced amino acid sequences are presentedin FIG. 1 (SEQ ID NO:1; SEQ ID NO:2).

To confirm the correct expression of the cloned gene, the N-terminalamino acid sequence of the native 22 kDa surface protein derived fromNeisseria meningitidis strain 608B was determined in order to compare itwith the amino 30 acid sequence deduced from the nucleotide sequencingdata. Outer membrane preparation derived from Neisseria meningitidisstrain 608B was resolved by electrophoresis on a 14% SDS-PAGE gel andtransferred onto a polyvinylidine difluoride membrane (MilliporeProducts, Bedford Mass.) according to a previously described method[Sambrook et al., Molecular Cloning; a laboratory manual, Cold SpringHarbor Laboratory Press (1989)]. The 22 kDa protein band was excisedfrom the gel and then subjected to Edman degradation using the AppliedBiosystems Inc. (Foster City, Calif.) model 473A automated proteinsequencer following the manufacturer's recommendations. The amino acidsequence E-G-A-S-G-F-Y-V-Q-A (SEQ ID NO: 32) corresponded to amino acids1-10 (SEQ ID NO:2) of the open reading frame, indicating that theNeisseria meningitidis strain 608B, 22 kDa surface protein has a 19amino acid leader peptide (amino acid residues −19 to −1 of SEQ IDNO:2).

A search of established databases confirmed that the Neisseriameningitidis strain 608B, 22 kDa surface protein (SEQ ID NO:2) or itsgene (SEQ ID NO:1) have not been described previously.

C. High Yield Expression And Purification of The Recombinant Neisseriameningitidis 22 kDa Surface Protein

The following process was developed in order to maximize the productionand purification of the recombinant Neisseria meningitidis 22 kDasurface protein expressed in Escherichia coli. This process is based onthe observation that the recombinant 22 kDa surface protein produced byEscherichia Coli strain BL21 (DE3) [Studier and Moffat, J. Mol. Biol.,189, p. 113 (1986)] carrying the plasmid pNP2202 can be found in largeamounts in the outer membrane, but can also be obtained from the culturesupernatant in which it is the most abundant protein. The culturesupernatant was therefore the material used to purify the recombinant 22kDa protein using affinity chromatography (FIG. 6A).

To generate an affinity chromatography matrix, monoclonal antibodiesMe-2, Me-3 and Me-5 (described in Example 2) were immobilized onCNBr-activated sepharose 4B (Pharmacia Biotech Inc., Piscataway, N.J.)according to the manufacturer's instructions.

To prepare the culture supernatant, an overnight culture of EscherichiaColi strain BL21 (DE3), harboring the plasmid pNP2202 was inoculated inLB broth (Gibco Laboratories, Grand Island, N.Y.) containing 25 mg/ml ofampicillin (Sigma) and was incubated 4 hours at 37° C. with agitation.The bacterial cells were removed from the culture media by twocentrifugations at 10,000×g for 10 minutes at 4° C. The culturesupernatant was filtered onto a 0.22 mm membrane (Millipore, Bedford,Mass.) and then concentrated approximately 100 times using anultra-filtration membrane (Amicon Co., Beverly, Mass.) with a molecularcut off of 10,000 Daltons. To completely solubilize the membranevesicles, EMPIGEN BB (Calbiochem Co., LaJolla, Calif.)) was added to theconcentrated culture supernatant to a final concentration of 1%(vol/vol). The suspension was incubated at room temperature for onehour, dialyzed overnight against several liters of 10 mM Tris-HClbuffer, pH 7.3 containing 0.05% EMPIGEN BB(vol/vol) and centrifuged at10,000×g for 20 minutes at 4° C. The antigen preparation was added tothe affinity matrix and incubated overnight at 4° C. with constantagitation. The gel slurry was poured into a chromatography column andwashed extensively with 10 mM Tris-HCl buffer, pH 7.3 containing 0.05%EMPIGEN BB (vol/vol). The recombinant 22 kDa protein was then elutedfrom the column with 1 M LiCl in 10 mM Tris-HCl buffer, pH 7.3. Thesolution containing the eluted protein was dialyzed extensively againstseveral liters of 10 mM Tris-HCl buffer, pH 7.3 containing 0.05% EMPIGENBB. Coomassie Blue and silver stained SDS-Page gels [Tsai and Frasch,Analytical Biochem., 119, pp. 19 (1982)] were used to evaluate thepurity of the recombinant 22 kDa surface protein at each step of thepurification process and representative results are presented in FIG.6A. Silver staining of the gels clearly demonstrated that thepurification process generated a fairly pure recombinant 22 kDa proteinwith only a very small quantity of Escherichia coli lipopolysaccharide.

The resistance to proteolytic cleavage of the purified recombinant 22kDa surface protein was also verified and the results are presented inFIG. 6B. Purified recombinant 22 kDa surface protein was treated asdescribed in Example 1 with a-chymotrypsin and trypsin at 2 mg per mg ofprotein and with 2 IU of proteinase K per mg of protein for 1 hour at37° C. with constant shaking. No reduction in the amount of protein wasobserved after any of these treatments. In comparison, partial orcomplete digestion depending on the enzyme selected was observed for thecontrol protein which was in this case bovine serum albumin (BSA,Sigma). Furthermore, longer periods of treatment did not result in anymodification of the protein. These latter results demonstrated thattransformed Escherichia coli cells can express the complete recombinant22 kDa surface protein and that this protein is also highly resistant tothe action of these three proteolytic enzymes as was the native proteinfound in Neisseria meningitidis. In addition, the purified recombinant22 kDa surface protein which is not embedded in the outer membrane ofEscherichia coli is still highly resistant to the action of theproteolytic enzymes.

We also verified the effect of the enzymatic treatments on the antigenicproperties of the recombinant 22 kDa protein. As determine by ELISA andWestern immunoblotting, the monoclonal antibodies described in Example 2readily recognized the recombinant 22 kDa surface protein that waspurified according to the process described above (FIG. 6C). Moreover,the reactivity of monoclonal antibody Me-5, as well as the reactivity ofother 22 kDa protein-specific monoclonal antibodies, with the purifiedrecombinant 22 kDa surface protein was not altered by any of the enzymetreatments, thus confirming that the antigenic properties of therecombinant 22 kDa protein seem similar to the ones described for thenative protein.

Important data were presented in Example 3 and can be summarized asfollows:

1) the complete nucleotide and amino acid sequences of the Neisseriameningitidis 22 kDa surface protein were obtained (SEQ ID NO:1; SEQ IDNO:2);

2) N-terminal sequencing of the native protein confirmed that theNeisseria meningitidis 22 kDa gene was indeed cloned;

3) this protein was not described previously;

4) it is possible to transform a host such as Escherichia coli andobtain expression of the recombinant Neisseria meningitidis 22 kDasurface protein in high yield;

5) it is possible to obtain the recombinant protein free of otherNeisseria meningitidis molecules and almost free of components producedby Escherichia coli;

6) the purified recombinant 22 kDa surface protein remains highlyresistant to the action of proteolytic enzymes such as a-chymotrypsin,trypsin and proteinase K; and

7) the antigenic properties of the recombinant 22 kDa protein compare tothe ones described for the native Neisseria meningitidis 22 kDa surfaceprotein.

Example 4 Molecular Conservation of the Gene Coding for the NeisseriaMeningitidis 22 kDa Surface Protein

To verify the molecular conservation among Neisseria isolates of thegene coding for the Neisseria meningitidis 22 kDa surface protein, a DNAdot blot hybridization assay was used to test different Neisseriaspecies and other bacterial species. First, the 525 base pair genecoding for the Neisseria meningitidis 22 kDa surface protein wasamplified by PCR, purified on agarose gel and labeled by random primingwith the non radioactive DIG DNA labeling and detection system(Boehringer Mannheim, Laval, Canada) following the manufacturer'sinstructions.

The DNA dot blot assay was done according to the manufacturer'sinstructions (Boehringer Mannheim). Briefly, the bacterial strains to betested were dotted onto a positively charge nylon membrane (BoehringerMannheim), dried and then treated as described in the DIG System'suser's guide for colony lifts. Pre-hybridizations and hybridizationswere done at 42° C. with solutions containing 50% formamide (Sigma). Thepre-hybridization solution also contained 100 mg/ml of denatured herringsperm DNA (Boehringer Mannheim) as an additional blocking agent toprevent non-specific hybridization of the DNA probe. The stringencywashes and detection steps using the chemiluminescent lumigen PPDsubstrate were also done as described in the DIG System's user's guide.

Stringency Washes

1. Wash the membranes twice for 5 min in ample 2×SSC, 0.1% SDS min atroom temperature with gentle agitation.

2. Transfer the membranes to 0.5×SSC, 0.1% SDS and wash twice for 15 minat 68° C. with gentle agitation.

For the 71 Neisseria meningitidis strains tested the results obtainedwith monoclonal antibody Me-7 and the 525 base pair DNA probe were inperfect agreement. According to the results, all the Neisseriameningitidis strains tested have the Neisseria meningitidis 22 kDasurface protein gene and they express the protein since they were allrecognized by the monoclonal antibody, thus confirming that this proteinis highly conserved among the Neisseria meningitidis isolates (Table 2).

The DNA probe also detected the gene coding for the Neisseriameningitidis 22 kDa surface protein in all Neisseria gonorrhoeae strainstested.

On the contrary, the monoclonal antibody Me-7 reacted only with 2 out ofthe 16 Neisseria gonorrhoeae strains tested indicating that the specificepitope is somehow absent, inaccessible or modified in Neisseriagonorrhoeae strains, or that most of the Neisseria gonorrhoeae strainsdo not express the protein even if they have the coding sequence intheir genome (Table 2).

A good correlation between the two detection methods was also observedfor Neisseria lactamica, since only one strain of Neisseria lactamicawas found to have the gene without expressing the protein (Table 2).This result could also be explained by the same reasons presented in thelast paragraph.

This may indicate that, although the 22 kDa is not expressed, or notaccessible on the surface of Neisseria gonorrhoeae strains, the 22 kDaprotein-coding gene of the Neisseria gonorrhoeae and Neisseria lactamicastrains may be used for construction of recombinant plasmids used forthe production of the 22 kDa surface protein or analogs. All suchprotein or analogs may be used for the prevention, detection, ordiagnosis of Neisseria infections. More particularly, such infectionsmay be selected from infections from Neisseria meningitidis, Neisseriagonorrhoeae, and Neisseria lactamica. Therefore, the 22 kDa surfaceprotein or analogs, may be used for the manufacture of a vaccine againstsuch infections. Moreover, the 22 kDa protein or analogs, may be usedfor the manufacture of a kit for the detection or diagnosis of suchinfections.

The results obtained with Moraxella catharralis strains showed that outof the 5 strains tested, 3 reacted with monoclonal antibody Me-7, butnone of them reacted with the DNA probe indicating that the gene codingfor the Neisseria meningitidis 22 kDa surface protein is absent from thegenome of these strains (Table 2).

Several other Neisserial species as well as other bacterial species (seefootnote, Table 2) were tested and none of them were found to bepositive by any of the two tests. This latter result seems to indicatethat the gene for the 22 kDa surface protein is shared only amongclosely related species of Neisseriacae.

TABLE 2 Reactivity of the 525 base pair DNA probe and monoclonalantibody Me-7 with different Neisseria species Number of strainsidentified by Neisseria species Monoclonal (number of strains tested)¹antibody Me-7 DNA probe Neisseria meningitidis (71) 71 71 Moraxellacatharallis (5) 3 0 Neisseria gonorrhoeae (16) 2 16 Neisseria lactamica(5) 4 5 ¹The following Neisserrial species and other bacterial specieswere also tested with the two assays and gave negative results: 1Neisseria cinerea, 1 Neisseria flava, 1 Neisseria flavescens, 2Neisseria mucosa, 4 Neisseria perflavalsicca,1 Neisseria perflava, 1 N.sicca, 1 N. subflava, 1 Alcaligenes feacalis (ATCC 8750), 1 Bordetellapertussis (9340), 1 Bordetella bronchiseptica, 1 Citrobacter freundii(ATCC 2080), 1 Edwarsiella tarda (ATCC 15947), 1 Enterobacter cloaca(ATCC 23355), 1 Enterobacter aerogenes (ATCC 13048), 1 Escherichia coli,1 Flavobacterium odoratum, 1 Haemophilus influenzae type b (Eaganstrain),1 Klebsiella pneumoniae (ATCC 13883), 1 Proteus rettgeri (ATCC25932), 1 Proteus vulgaris (ATCC #13315), 1 Pseudomonas aeruginosa (ATCC9027), 1 Salmonella typhimurium (ATCC 14028), 1 Serrati marcescens (ATCC8100), 1 Shigella flexneri (ATCC 12022), 1 Shigella sonnei (ATCC 9290),and 1 Xanthomonas maltophila.

In conclusion, the DNA hybridization assay clearly indicated that thegene coding for the Neisseria meningitidis 22 kDa surface protein ishighly conserved among the pathogenic Neisseria. Furthermore, theresults obtained clearly showed that this DNA probe could become avaluable tool for the rapid and direct detection of pathogenic Neisseriabacteria in clinical specimen. This probe could even be refined todiscriminate between the Neisseria meningitidis and Neisseriagonorrhoeae.

Example 5 Bacteriolytic and Protective Properties of the MonoclonalAntibodies

The bacteriolytic activity of the purified Neisseria meningitidis 22 kDasurface protein-specific monoclonal antibodies was evaluated in vitroaccording to a method described previously [Brodeur et al., Infect.Immun., 50, p. 510 (1985); Martin et al., Infect. Immun., 60, p. 2718(1992)]. In the presence of a guinea pig serum complement, purifiedmonoclonal antibodies Me-I and Me-7 efficiently killed Neisseriameningitidis strain 608B. Relatively low concentrations of each of thesemonoclonal antibodies reduced by more than 50% the number of viablebacteria. The utilization of higher concentrations of purifiedmonoclonal antibodies Me-1 and Me-7 resulted in a sharp decrease (up to99%) in the number of bacterial colony forming units. Importantly, thebacteriolytic activity of these monoclonal antibodies is complementdependent, since heat-inactivation of the guinea pig serum for 30minutes at 56° C. completely abolished the killing activity. The othermonoclonal antibodies did not exhibit significant bacteriolytic activityagainst the same strain. The combined, representative results of severalexperiments are presented in FIG. 7, wherein the results shown for Me-7are representative and consistent with the results obtained for Me-1.The results shown for Me-2 are representative and consistent with theresults obtained for the other monoclonal antibodies Me-3, Me-5 andMe-6.

A mouse model of infection, which was described previously by one of theinventors [Brodeur et al, Infect. Immun., 50, p. 510 (1985); Brodeur etal., Can. J. Microbial., 32, p. 33 (1986)] was used to assess theprotective activity of each monoclonal antibody. Briefly, Balb/c micewere injected intraperitoneally with 600 ml of ascitic fluid containingthe monoclonal antibodies 18 hours before the bacterial challenge. Themice were then challenged with one ml of a suspension containing 1000colony forming units of Neisseria meningitidis strain 608B, 4% mucin(Sigma) and 1.6% hemoglobin (Sigma). The combined results of severalexperiments are presented in Table 3. It is important to note that onlythe bacteriolytic monoclonal antibodies Me-1 and Me-7 protected the miceagainst experimental Neisseria meningitidis infection. Indeed, theinjection of ascitic fluid containing these two monoclonal antibodiesbefore the bacterial challenge significantly increased the rate ofsurvival of Balb/c mice to 70% or more compared to the 9% observed inthe control groups receiving either 600 ml Sp2/0 induced ascitic fluidor 600 ml ascitic fluid containing unrelated monoclonal antibodies.Results have also indicated that 80% of the mice survived the infectionif they were previously injected with 400 μg of protein A purified Me-718 hours before the bacterial challenge. Subsequent experiments arepresently being done to determine the minimal antibody concentrationnecessary to protect 50% of the mice. Lower survival rates from 20 to40% were observed for the other Neisseria meningitidis 22 kDa surfaceprotein-specific monoclonal antibodies.

TABLE 3 Evaluation of the immunoprotective potential of the 22 kDasurface protein-specific monoclonal antibodies against Neisseriameningitidis strain 608B (B:2a:P1.2) Monoclonal Number of living miceafter challenge Antibodies 24 hr 72 hrs % of survival Me-1 29/30 23/30 76 Me-2 17/20 3/20 25 Me-3  5/10 2/10 20 Me-5 11/20 8/20 40 Me-7 10/107/10 70 purified Me-7 13/15 12/15  80 Control  31/100  9/100 9

In conclusion, the results clearly indicated that an antibody specificfor the Neisseria meningitidis 22 kDa surface protein can efficientlyprotect mice against an experimental lethal challenge. The induction ofprotective antibodies by an antigen is one of the most importantcriteria to justify further research on potential vaccine candidate.

Example 6 Immunization with Purified Recombinant 22 kDa Surface ProteinConfers Protection Against Subsequent Bacterial Challenge

Purified recombinant 22 kDa surface protein was prepared according tothe protocol presented in Example 3, and was used to immunize Balb/cmice to determine its protective effect against challenge with a lethaldose of Neisseria meningitidis 608B (B:2a:P1.2). It was decided to usethe purified recombinant protein instead of the native meningococcalprotein in order to insure that there was no other meningococcal antigenin the vaccine preparation used during these experiments. The mousemodel of infection used in these experiments was described previously byone of the inventors [Brodeur et al., Infec. Immun., 50, p. 510 (1985);Brodeur et al., Can. J. Microbiol., 32, p. 33 (1986)]. The mice wereeach injected subcutaneously three times at three-week intervals with100 ml of the antigen preparation containing either 10 or 20 μg permouse of the purified recombinant 22 kDa surface protein. QuilA was theadjuvant used for these experiments at a concentration of 25 μg perinjection. Mice in the control groups were injected following the sameprocedure with either 10 or 20 μg of BSA, 20 μg of concentrated culturesupernatant of Escherichia coli strain BL21(DE3) carrying the plasmidpWKS30 without the insert gene for the meningococcal protein prepared asdescribed in Example 3, or phosphate-buffered saline. Serum samples fromeach mouse were obtained before each injection in order to analyze thedevelopment of the immune response against the recombinant protein. Twoweeks following the third immunization the mice in all groups wereinjected intraperitoneally with 1 ml of a suspension containing 1000colony forming units of Neisseria meningitidis strain 608B in 4% mucin(Sigma) and 1.6% hemoglobin (Sigma).

The results of these experiments are presented in Table 4. Eightypercent (80%) of the mice immunized with the purified recombinant 22 kDasurface protein survived the bacterial challenge compared to 0 to 42% inthe control groups. Importantly, the mice in the control group injectedwith concentrated Escherichia Coli culture supernatant were notprotected against the bacterial challenge. This latter result clearlydemonstrated that the components present in the culture media and theEscherichia Coli antigens that might be present in small amounts afterpurification do not contribute to the observed protection againstNeisseria meningitidis.

TABLE 4 Immunization With Purified Recombinant 22 kDa Surface ProteinConfers Protection Against Subsequent Bacterial Challenge with Neisseriameningitidis 608B (B:2a:P1.2) strain. Number of living mice afterchallenge % of Experiment Group 24 h 48 h 72 h survival 1 10 μg ofpurified 20/20  16/20  80 22 kDa protein 10 μg of BSA 17/19  8/19 42 220 μg of purified 9/10 8/10 8/10 80 22 kDa protein 20 μg of 7/10 5/102/10 20 concentrated E. coli supernatant 20 μg of BSA 6/10 4/10 2/10 20Phosphate 8/10 0/10 0/10 0 buffered saline

Conclusion

The injection of purified recombinant 22 kDa surface protein greatlyprotected the immunized mice against the development of a lethalinfection by Neisseria meningitidis.

Antibodies according to this invention are exemplified by murinehybridoma cell lines producing monoclonal antibodies Me-1 and Me-7deposited in the American Type Culture Collection in Rockville, Md., USAon Jul. 21, 1995. The deposits were assigned accession numbers HB 11959(Me-1) and HB 11958 (Me-7).

Example 7 Sequence Analysis of Other Strains of Neisseria meningitidisand of Neisseria gonorrhoeae

The 2.75 kb c/al digested DNA fragment containing the gene coding forthe 22 kDa surface protein was isolated from the genomic DNA of thedifferent strains of Neisseria meningitidis and Neisseria gonorrhoeae asdescribed in Example 3.

a) MCH88 strain: The nucleotide sequence of strain MCH88 (clinicalisolate) is presented in FIG. 8 (SEQ ID NO:3). From experimentalevidence obtained from strain 608B (Example 3), a putative leadersequence was deduced corresponding to amino acid −19 to −1(M-K-K-A-L-A-A-L-I-A-L-A-L-P-A-A-A-L-A) (SEQ ID NO: 33). A search ofestablished databases confirmed that 22 kDa surface protein fromNeisseria meningitidis strain MCH 188 (SEQ ID NO:4) or its gene (SEQ IDNO:3) have not been described previously.

b) Z4063 strain: The nucleotide sequence of strain Z4063 (Wang J.-F. etal. Infect. Immun., 60, p. 5267 (1992)) is presented in FIG. 9 (SEQ IDNO:5). From experimental evidence obtained from strain 608B (Example 3),a putative leader sequence was deduced corresponding to amino acid −19to −1 (M-K-K-A-L-A-T-L-I-A-L-A-L-P-A-A-A-L-A) (SEQ ID NO: 34). A searchof established databases confirmed that 22 kDa surface protein fromNeisseria meningitidis strain Z4063 (SEQ ID NO:6) or its gene (SEQ IDNO:5) have not been described previously.

c) Neisseria gonorrhoeae strain b2: The nucleotide sequence of Neisseriagonorrhoeae strain b2 (serotype 1. Nat. Ref. Center for Neisseria, LCDC,Ottawa, Canada) is described in FIG. 10 (SEQ ID NO:7). From experimentalevidence obtained from strain 608B (Example 3), a putative leadersequence was deduced corresponding to amino acid −19 to −1(M-K-K-A-L-A-A-L-I-A-L-A-L-P-A-A-A-L-A)(SEQ ID NO: 33). A search ofestablished databases confirmed that 22 kDa surface protein fromNeisseria gonorrhoeae strain b2 (SEQ ID NO:8) or its gene (SEQ ID NO:7)have not been described previously.

FIG. 11 shows the consensus sequence established from the DNA sequenceof all four strains tested. The MCH88 strain showed an insertion of onecodon (TCA) at nucleotide 217, but in general the four strains showedstriking homology.

FIG. 12 depicts the homology between the deduced amino acid sequenceobtained from the four strains. There is greater than 90% identitybetween all four strains.

Example 8 Immunological Response of Rabbits and Monkeys to the 22 kDaNeisseria meningitidis Surface Protein

Rabbits and monkeys were immunized with the recombinant 22 kDa proteinto assess the antibody response in species other than the mouse.

a) Rabbits

Male New Zealand rabbits were immunized with outer membrane preparationsobtained from E. coli strain JM109 with the plasmid pN₂₂O₂ or with thecontrol plasmid pWKS30 (the strain and the plasmids are described inExample 3). The lithium chloride extraction used to obtain these outermembrane preparations was performed in a manner previously described bythe inventors [Brodeur et al, Infect. Immun. 50, 510 (1985)]. Theprotein content of these preparations was determined by the Lowry methodadapted to membrane fractions [Lowry et al, J. Biol. Chem. 193, 265(1951)]. The rabbits were injected subcutaneously and intramuscularly atseveral sites twice at three week intervals with 150 μg of one of theouter membrane preparations described above. QuilA, at a finalconcentration of 20% (vol./vol.) (CedarLane Laboratories, Hornby, Ont.,Canada), was the adjuvant used for these immunizations. The developmentof the specific humoral response was analyzed by ELISA using outermembrane preparations extracted from Neisseria meningitidis strain 608B(B:2a:P1.2) as coating antigen and by Western immunoblotting followingmethods already described by the inventors [Brodeur et al., Infect.Immun. 50, 510 (1985); Martin et al, Eur. J. Immunol. 18, 601 (1988)].Alkaline phosphatase or peroxidase-labeled Donkey anti-rabbitimmunoglobulins (Jackson ImmunoResearch Laboratories, West Grove, Pa.)were used for these assays.

The injection of E. coli outer membrane preparation containing the 22kDa recombinant protein in combination with QuilA adjuvant induced inthe rabbit a strong specific humoral response of 1/32,000 as determinedby ELISA (FIG. 13). The antibodies induced after the injection of therecombinant 22 kDa protein reacted with the purified recombinant 22 kDaprotein, but more importantly they also recognized the native protein asexpressed, folded and embedded in the outer membrane of Neisseriameningitidis. Western Immunoblotting experiments clearly indicated thatthe antibodies present after the second injection recognized onnitrocellulose membrane the same protein band as the one revealed by MabMe-2 (described in Example 2), which is specific for the 22 kDa protein.

b) Monkeys

Two Macaca fascicularis (cynomolgus) monkeys were respectively immunizedwith two injections of 100 μg (K28) and 200 μg (1276) of affinitypurified recombinant 22 kDa protein per injection. The methods used toproduce and purify the protein from E. coli strain BL2IDe3 weredescribed in Example 3. Alhydrogel, at a final concentration of 20%(vol./vol.) (CedarLane Laboratories, Hornby, Ont., Canada), was theadjuvant used for these immunizations. The monkeys received twointramuscular injections at three weeks interval. A control monkey (K65)was immunized with an unrelated recombinant protein preparationfollowing the same procedures. The sera were analyzed as describedabove. Alkaline phosphatase or peroxidase-labeled Goat anti-humanimmunoglobulins (Jackson ImmunoResearch Laboratories, West Grove, Pa.)were used for these assays.

The specific antibody response of monkey K28 which was immunized with100 μg of purified protein per injection appeared faster and wasstronger than the one observed for monkey 1276 which was injected with200 μg of protein (FIG. 14). Antibodies specific for the native 22 kDaprotein as detected by Western immunoblotting were already present inthe sera of the immunized monkeys twenty one days after the firstinjection, but were absent in the sera of the control monkey after twoinjections of the control antigen.

Conclusion

The data presented in Examples 2 and 5 clearly showed that the injectionof the recombinant 22 kDa protein can induce a protective humoralresponse in mice which is directed against Neisseria meningitidisstrains. More importantly, the results presented in this exampledemonstrate that this immunological response is not restricted to onlyone species, but this recombinant surface protein can also stimulate theimmune system of other species such as rabbit or monkey.

Example 9 Epitope Mapping of the 22 kDa Neisseria meningitidis Protein

Neisseria meningitidis 22 kDa surface protein was epitope mapped using amethod described by one of the inventors [Martin et al. Infect. Immun(1991): 59:1457-1464]. Identification of the linear epitopes wasaccomplished using 18 overlapping synthetic peptides covering the entireNeisseria meningitidis 22 kDa protein sequence derived from strain 608B(FIG. 15) and hyperimmune sera obtained after immunization with thisprotein. The identification of immunodominant portions on the 22 kDaprotein may be helpful in the design of new efficient vaccines.Furthermore, the localization of these B-cell epitopes also providesvaluable information about the structural configuration of the proteinin the outer membrane of Neisseria meningitidis.

All peptides were synthesized by BioChem Immunosystems Inc. (Montreal,Canada) with the Applied Biosystems (Foster City, Calif.) automatedpeptide synthesizer. Synthetic peptides were purified by reverse-phasehigh-pressure liquid chromatography. Peptides CS-845, CS-847, CS-848,CS-851, CS-852 and CS-856 (FIG. 15) were solubilized in a small volumeof 6M guanidine-HCl (J. T. 15 Baker, Ontario, Canada) or dimethylsulfoxide (J. T. Baker). These peptides were then adjusted to 1 mg/mlwith distilled water. All the other peptides were freely soluble indistilled water and were also adjusted to 1 mg/ml.

Peptide enzyme-linked immunosorbent assays (ELISA) were performed bycoating synthetic peptides onto microtitration plates (Immulon 4,Dynatech Laboratories Inc., Chantilly, Va.) at a concentration of 50μg/ml in 50 mM carbonate buffer, pH 9.6. After overnight incubation atroom temperature, the plates were washed with phosphate-buffered saline(PBS) containing 0.05% (wt/vol) Tween 20 (Sigma Chemical Co., St.-Louis,Mo.) and blocked with PBS containing 0.5% (wt/vol) bovine serum albumin(Sigma). Sera obtained from mice and monkeys immunized with affinitypurified recombinant 22 kDa surface protein were diluted and 100 μl perwell of each dilution were added to the ELISA plates and incubated for 1h at 37° C. The plates were washed three times, and 100 μl of alkalinephosphatase-conjugated goat anti-mouse or anti-human immunoglobulins(Jackson ImmunoResearch Laboratories, West Grove, Pa.) diluted accordingto the manufacturer's recommendations was added. After incubation for 1h at 37° C., the plates were washed and 100 μl of diethanolamine (10%(vol/vol), pH 9.8) containing p-nitro-phenylphosphate (Sigma) at 1 mg/mlwas added. After 60 min., the reaction (λ=k=410 nm) was readspectrophotometrically with a microplate reader.

Mouse and monkey antisera obtained after immunization with affinitypurified recombinant 22 kDa protein (Example 8) were successfully usedin combination with eighteen overlapping synthetic peptides to localizeB-cell epitopes on the protein. These epitopes are clustered withinthree antigenic domains on the protein.

The first region is located between amino acid residues 51 and 86.Computer analysis using different algorithms suggested that this regionhas the highest probability of being immunologically important since itis hydrophilic and surface exposed. Furthermore, comparison of the fourprotein sequences which is presented in FIG. 12 indicates that one ofthe major variation, which is the insertion of one amino acid residue atposition 73, is also located in this region.

The antisera identified a second antigenic domain located between aminoacid residues 110 and 140. Interestingly, the sequence analysis revealedthat seven out of the fourteen amino acid residues that are notconserved among the four protein sequences are clustered within thisregion of the protein.

A third antigenic domain located in a highly conserved portion of theprotein, between amino acid residues 31 and 55, was recognized only bythe monkeys' sera.

Example 10 Heat-Inducible Expression Vector for the Large ScaleProduction of the 22 kDa Surface Protein

The gene coding for the Neisseria meningitidis 22 kDa surface proteinwas inserted into the plasmid p629 [George et al. Bio/technology 5:600-603 (1987)]. A cassette of the bacteriophage λ cl857 temperaturesensitive repressor gene, from which the functional Pr promoter has beendeleted, is carried by the plasmid p629 that uses the PL promoter tocontrol the synthesis of the 22 kDa surface protein. The inactivation ofthe cl857 repressor by a temperature shift from 30° C. to temperaturesabove 38° C. results in the production of the protein encoded by theplasmid. The induction of gene expression in E. coli cells by atemperature shift is advantageous for large scale fermentation since itcan easily be achieved with modern fermentors. Other inducibleexpression vectors usually require the addition of specific moleculeslike lactose or isopropylthio-β-D-galactoside (IPTG) in the culturemedia in order to induce the expression of the desired gene.

A 540 nucleotide fragment was amplified by PCR from the Neisseriameningitidis strain 608B genomic DNA using the following twooligonucleotide primers (SEQ ID NOS: 27 & 28, respectively) (OCRR8:5′-TAATAGATCTATGAAAAAAGCACTTGCCAC-3′ and OCRR9:3′-CACGCGCAGTTTAAGACTTCTAGATTA-5′). These primers correspond to thenucleotide sequences found at both ends of the 22 kDa gene. To simplifythe cloning of the PCR product, a Bgl II (AGATCT) restriction site wasincorporated into the nucleotide sequence of these primers. The PCRproduct was purified on agarose gel before being digested with Bgl II.This Bgl II fragment of approximately 525 base pairs was then insertedinto the Bgl II and Bam HI sites of the plasmid p629. The plasmidcontaining the PCR product insert named pNP2204 was used to transform E.coli strain DH5αF'IQ. A partial map of the plasmid pNP2204 is presentedin FIG. 16. The resulting colonies were screened with Neisseriameningitidis 22 kDa surface-protein specific monoclonal antibodiesdescribed in Example 2. Western blot analysis of the resulting clonesclearly indicated that the protein synthesized by E. Coli was completeand migrated on SDS-PAGE gel like the native Neisseria meningitidis 22kDa surface protein. Plasmid DNA was purified from the selected cloneand then sequenced. The nucleotide sequence of the insert present in theplasmid perfectly matched the nucleotide sequence of the gene coding forthe Neisseria meningitidis 22 kDa protein presented in FIG. 1.

To study the level of synthesis of the 22 kDa surface protein, thetemperature-inducible plasmid pNP2204 was used to transform thefollowing E. coli strains: W3110, JM105, BL21, TOPP1, TOPP2 and TOPP3.The level of synthesis of the 22 kDa surface protein and thelocalization of the protein in the different cellular fractions weredetermined for each strain. Shake flask cultures in LB broth (Gibco BRL,Life Technologies, Grand Island, N.Y.) indicated that a temperatureshift from 30° C. to 39° C. efficiently induced the expression of thegene. Time course evaluation of the level of synthesis indicated thatthe protein appeared, as determined on SDS-PAGE gel, as soon as 30 minafter induction and that the amount of protein increased constantlyduring the induction period. Expression levels between 8 to 10 mg of 22kDa protein per liter were determined for E. coli strains W3110 andTOPP1. For both strains, the majority of the 22 kDa protein isincorporated in the bacterial outer membrane.

Example 11 Purification of the Neisseria meningitidis 22 kDa Protein

Since the vast majority of the 22 kDa protein is found embedded in theouter membrane of E. coli strains, the purification protocol presentedin this Example is different from the one already described in Example 3where a large amount of protein was released in the culture supernatant.An overnight culture incubated at 30° C. of either E. coli strain W3110or TOPP1 harboring the plasmid pNP2204 was inoculated in LB brothcontaining 50 μg/ml of Ampicillin (Sigma) and was grown at 30° C. withagitation (250 rpm) until it reached a cell density of 0.6 (λ=600 nm),at which point the incubation temperature was shifted to 39° C. forthree to five hours to induce the production of the protein. Thebacterial cells were harvested by centrifugation at 8,000×g for 15minutes at 4° C. and washed twice in phosphate buffered saline (PBS), pH7.3. The bacterial cells were ultrasonically broken (ballisticdisintegration or mechanical disintegration with a French press may alsobe used). Unbroken cells were removed by centrifugation at 5,000×g for 5minutes and discarded. The outer membranes were separated fromcytoplasmic components by centrifugation at 100,000 xg for 1 h at 10° C.The membrane-containing pellets were resuspended in a small volume ofPBS, pH 7.3. To solubilize the 22 kDa surface protein from themembranes, detergents such as EMPIGEN BB (Calbiochem Co., LaJolla,Calif.), Zwittergent-3,14 (Calbiochem Co.), or β-octyglucoside (Sigma)were used. The detergent was added to the membrane fraction at finalconcentration of 3% and the mixture was incubated for 1 h at 20° C. Thenon soluble material was removed by centrifugation at 100,000 xg for 1 hat 10° C.

The 22 kDa protein was efficiently solubilized by either three of thedetergents, however β-octylglucoside had the advantage of easilyremoving several unwanted membrane proteins since they were notsolubilized and could be separated from the supernatant bycentrifugation. To remove the detergent, the 22 kDa containingsupernatant was dialyzed extensively against several changes of PBSbuffer. Proteinase K treatment (as in Example 1) can be used to furtherremove unwanted proteins from the 22 kDa surface protein preparation.Differential precipitation using ammonium sulfate or organic solvents,and ultrafiltration are two additional steps that can be used to removeunwanted nucleic acid and lipopolysaccharide contaminants from theproteins before gel permeation and ion-exchange chromatography can beefficiently used to obtain the purified 22 kDa protein. Affinitychromatography, as described in Example 3, can also be used to purifythe 22 kDa protein.

Example 12 Use of 22 kDa Surface Protein as a Human Vaccine

To formulate a vaccine for human use, appropriate 22 kDa surface proteinantigens may be selected from the polypeptides described herein. Forexample, one of skill in the art could design a vaccine around the 22kDa polypeptide or fragments thereof containing an immunogenic epitope.The use of molecular biology techniques is particularly well-suited forthe preparation of substantially pure recombinant antigens.

The vaccine composition may take a variety of forms. These include, forexample, solid, semi-solid, and liquid dosage forms, such as powders,liquid solutions or suspensions, and liposomes. Based on our belief thatthe 22 kDa surface protein antigens of this invention may elicit aprotective immune response when administered to a human, thecompositions of this invention will be similar to those used forimmunizing humans with other proteins and polypeptides, e.g., tetanusand diphtheria. Therefore, the compositions of this invention willpreferably comprise a pharmaceutically acceptable adjuvant such asincomplete Freund's adjuvant, aluminum hydroxide, a muramyl peptide, awater-in-oil emulsion, a liposome, an ISCOM or CTB, or a non-toxic Bsubunit form cholera toxin. Most preferably, the compositions willinclude a water-in-oil emulsion or aluminum hydroxide as adjuvant.

The composition would be administered to the patient in any of a numberof pharmaceutically acceptable forms including intramuscular,intradermal, subcutaneous or topic. Preferably, the vaccine will beadministered intramuscularly.

Generally, the dosage will consist of an initial injection, mostprobably with adjuvant, of about 0.01 to 10 mg, and preferably 0.1 to1.0 mg of 22 kDa surface protein antigen per patient, followed mostprobably by one or more booster injections. Preferably, boosters will beadministered at about 1 and 6 months after the initial injection.

A consideration relating to vaccine development is the question ofmucosal immunity. The ideal mucosal vaccine will be safely taken orallyor intranasally as one or a few doses and would elicit protectiveantibodies on the appropriate surfaces along with systemic immunity. Themucosal vaccine composition may include adjuvants, inert particulatecarriers or recombinant live vectors.

The anti-22 kDa surface protein antibodies of this invention are usefulfor passive immunotherapy and immunoprophylaxis of humans infected withNeisseria meningitidis or related bacteria such as Neisseria gonorrhoeaeor Neisseria lactamica. The dosage forms and regimens for such passiveimmunization would be similar to those of other passive immunotherapies.

An antibody according to this invention is exemplified by a hybridomaproducing MAbs Me-1 or Me-7 deposited in the American Type CultureCollection in Rockville, Md., USA on Jul. 21, 1995, and identified asMurine Hybridoma Cell Lines, Me-1 and Me-7 respectively. These depositswere assigned accession numbers HB 11959 (Me-1) and HB 11958 (Me-7).

While we have described herein a number of embodiments of thisinvention, it is apparent that our basic embodiments may be altered toprovide other embodiments that utilize the compositions and processes ofthis invention. Therefore, it will be appreciated that the scope of thisinvention includes all alternative embodiments and variations that aredefined in the foregoing specification and by the claims appendedthereto; and the invention is not to be limited by the specificembodiments which have been presented herein by way of example.

1. A method of isolating a polypeptide comprising: a) isolating aculture of Neisseria meningitidis bacteria; and b) isolating an outermembrane portion from the culture of the bacteria, wherein thepolypeptide comprises (i) an amino acid sequence at least 90% identicalto the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQID NO:6, wherein the polypeptide is capable of eliciting an antibodythat specifically binds to a protein consisting of the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; or (ii)the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:6.
 2. The method according to claim 1 further comprising isolatingthe polypeptide from the outer membrane portion.
 3. The method of claim1, further comprising treating the outer membrane portion withproteinase K.
 4. The method of claim 1 wherein the polypeptide issubstantially purified from other N. meningitidis proteins.
 5. Themethod of claim 1 wherein the polypeptide is a recombinant polypeptide.6. The method of claim 1 wherein the polypeptide is capable of inducingan immunological response to N. meningitidis.
 7. The method of claim 1wherein the polypeptide comprises a polypeptide fragment of the aminoacid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:4, or SEQID NO:6, wherein the polypeptide fragment has at least one immunogenicepitope, and wherein the polypeptide fragment is capable of eliciting anantibody that specifically binds to a protein consisting of the sequenceset forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQID NO:8.
 8. The method according to claim 7 wherein the polypeptidefragment comprises the amino acid sequence set forth at (a) residue 31to residue 55 of SEQ ID NO:2; (b) residue 51 to residue 86 of SEQ IDNO:2; or (c) residue 110 to residue 140 of SEQ ID NO:2.
 9. The methodaccording to claim 7 wherein the polypeptide fragment comprises theamino acid sequence set forth in any one of SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, and SEQ ID NO:
 26. 10. A method of manufacturing a vaccinecomprising (a) isolating the polypeptide according to the method ofclaim 1 or claim 7; and (b) formulating the polypeptide with apharmaceutically acceptable excipient.
 11. The method according to claim10 further formulating the polypeptide with a pharmaceuticallyacceptable adjuvant.
 12. The method according to claim 10 wherein thevaccine is formulated in a suitable vehicle.
 13. A method for producinga recombinant polypeptide wherein the recombinant polypeptide comprises(a) the amino acid sequence set forth from amino acid residue 31 toamino acid residue 55 of SEQ ID NO:2; (b) the amino acid sequence setforth from amino acid residue 51 to amino acid residue 86 of SEQ IDNO:2; or (c) the amino acid sequence set forth from amino acid residue110 to amino acid residue 140 of SEQ ID NO:2, said method comprisingculturing a host cell that comprises an expression vector, wherein theexpression vector comprises a polynucleotide that encodes therecombinant polypeptide, and wherein the polynucleotide is operativelylinked to one or more expression control sequences.
 14. The methodaccording to claim 13 wherein the recombinant polypeptide at least 90%identical to the amino acid sequence set forth in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, or SEQ ID NO:8.
 15. The method according to claim 13wherein the recombinant polypeptide is capable of inducing animmunological response against Neisseria.
 16. The method according toclaim 13 wherein the recombinant polypeptide is capable of eliciting anantibody that specifically binds to a protein consisting of the aminoacid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQID NO:8.
 17. The method according to claim 13 wherein the host cell is aeukaryotic cell or a bacteria cell.
 18. The method according to claim 13wherein the host cell is a bacterial cell.
 19. The method according toclaim 13 wherein the one or more expression control sequences isheterologous.
 20. The method according to claim 18 further comprising(a) isolating a culture of the bacterial cell; and (b) isolating anouter membrane portion from said bacterial cell culture.
 21. The methodaccording to claim 20 further comprising isolating the recombinantpolypeptide from the outer membrane portion.
 22. A method ofmanufacturing a vaccine comprising (a) producing a recombinantpolypeptide according to the method of claim 13; and (b) formulating therecombinant polypeptide with a pharmaceutically acceptable excipient.